WO2024086999A1

WO2024086999A1 - A method for regulating migrasome formation and/or migrasome-mediated biological process

Info

Publication number: WO2024086999A1
Application number: PCT/CN2022/127174
Authority: WO
Inventors: Li Yu; Tianlun DING
Original assignee: Tsinghua University
Priority date: 2022-10-25
Filing date: 2022-10-25
Publication date: 2024-05-02

Abstract

Provided is a method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of PIP2 related pathway in said cell.

Description

A METHOD FOR REGULATING MIGRASOME FORMATION AND/OR MIGRASOME-MEDIATED BIOLOGICAL PROCESS

BACKGROUND OF THE INVENTION

Migrasomes are recently discovered organelles of migratory cells. During migration, retraction fibers are pulled out of the trailing edge of cells, and migrasomes grow at the branch points or the ends of these retraction fibers. Eventually, when cells migrate away, the retraction fibers break and migrasomes are left behind. Migrasomes play important roles in various biological processes; for example, during zebrafish embryonic development, migrasomes enriched with the chemokine CXCL12 are concentrated in the embryonic shield cavity, where CXCL12 works as a chemoattractant to guide the migration of dorsal forerunner cells. Thus, migrasomes play an important role in organ morphogenesis. In addition, migrasomes have been shown to mediate lateral transfer of mRNA among cells.

Migrasomes have been observed in various biological settings and have been shown to play important physiological roles in vivo. However, the regulating of migrasomes is less clear.

SUMMARY OF THE INVENTION

The present disclosure provides a method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of PIP ₂ in said cell.

The present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of PIP ₂ therein.

The present disclosure provides a method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of PIP5K1 in said cell.

The present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of PIP5K1 therein.

The present disclosure provides a method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of Rab35 in said cell.

The present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of Rab35 therein.

The present disclosure provides a method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of integrin α5 (ITGa5) in said cell.

The present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of ITGa5 therein.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCES

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1A-1K illustrate Generation of PIP ₂ by PIP5K1A at the migrasome formation sites. (A) Live-cell SIM images of NRK cells expressing PH-GFP and TSPAN4-mCherry. Green, PH; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (B) Immunofluorescence (IF) staining of PIP ₂ in an NRK cell line overexpressing TSPAN4-mCherry. Cells were imaged by confocal microscopy. Green, PIP ₂; red, TSPAN4; yellow, merge. Scale bar, 10 μm.Boxed regions are enlarged at the right. (C) Time-lapse imaging of NRK cells stably expressing PH-GFP and TSPAN4-mCherry. Images were taken every 10 min by SIM. Green, PH; red, TSPAN4; yellow, merge. Scale bar, 2 μm. (D) Time-lapse imaging of NRK cells stably expressing PH-TagBFP, TSPAN4-GFP and ITGα5-mCherry. Confocal microscopy images were taken every 1 min. Cyan, PH; yellow, TSPAN4; red, ITGα5; white, merge. Scale bar, 2 μm. White arrowheads indicate two migrasome formation sites. (E) Statistical analysis of normalized fluorescence intensity of PH, TSPAN4 and ITGα5 at migrasome formation sites during migrasome formation. Mean±s.e.m., n=10 from 4 independent cells. (F) Immunofluorescence (IF) staining of PIP5K1A in the NRK cell line overexpressing TSPAN4-mCherry. Cells were imaged by confocal microscopy. Green, PIP5K1A; red, TSPAN4; yellow, merge. Scale bar, 10 μm. (G) Time-lapse imaging of NRK cells stably expressing PIP5K1A-GFP and TSPAN4-mCherry. Images were taken every 7 min by SIM. Green, PIP5K1A; red, TSPAN4; yellow, merge. Scale bar, 2 μm. (H) Live-cell images of NRK cells expressing TSPAN4-GFP under treatment with ISA2011B or DMSO (control) . Cells were imaged by confocal microscopy. Green, TSPAN4. Scale bar, 10 μm. (I) Statistical analysis of the number of migrasomes/100 μm retraction fiber per cell. The original images were captured as in H. n=54 for control; n=64 for ISA011B treatment. Mean±s.e.m., unpaired t-test. (J) Live-cell confocal microscopy images of WT NRK-TSPAN4-mCherry cells and PIP5K1A-KO NRK-TSPAN4-mCherry cells without and with stable expression of GFP-PIP5K1A (WT) , (D306A) , (L199I) and (L207I) . Green, PIP5K1A; red, TSPAN4; yellow, merge. Scale bar, 10 μm. (K) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in J. n=68 for WT; n=61 for PIP5K1A KO; n=65 for PIP5K1A-WT rescue; n=66 for PIP5K1A D306A rescue; n=60 for PIP5K1A L199I rescue; n=63 for PIP5K1A L207I rescue. Mean±s.e.m., unpaired t-test.

FIG. 2A-2J illustrate PIP ₂ recruits Rab35 to migrasome formation sites. (A) MA plot of PIP ₂-binding proteins in migrasomes compared to cell bodies. Rab35 is highlighted in red. (B) Immunofluorescence (IF) staining of Rab35 in NRK cells expressing TSPAN4-mCherry. Cells were imaged by confocal microscopy. Green, Rab35; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (C) Live-cell SIM images of NRK cells stably expressing TSPAN4-GFP and mCherry-Rab35. Green, TSPAN4; red, Rab35; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (D) Time-lapse imaging of NRK cells stably expressing TSPAN4-GFP and mCherry-Rab35. Images were taken every 10 min by SIM. Green, TSPAN4; red, Rab35; yellow, merge. Scale bar, 2 μm. (E) Live-cell SIM images of NRK cells stably expressing TSPAN4-GFP and mCherry-Rab35. Cells were treated without (Ctrl) or with ISA2011B for 8 h. Green, TSPAN4; red, Rab35; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the bottom. (F) Statistical analysis of the number of Rab35 puncta per 100 μm retraction fiber per cell. The original images were captured as in E. n=36 for control; n=23 for ISA2011B treatment. Mean±s.e.m., unpaired t-test. (G) Live-cell SIM images of WT or PIP5K1A-KO NRK cells stably expressing GFP-Rab35 and TSPAN4-mCherry. Green, Rab35; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Enlarged images are shown underneath. (H) Statistical analysis of the number of Rab35 puncta per 100 μm retraction fiber per cell. The original images were captured as in G. n=21 for WT; n=16 for PIP5K1A KO. Mean±s.e.m., unpaired t-test. (I) Live-cell SIM images of NRK cells stably expressing GFP-Rab35-7Glu and TSPAN4-mCherry. Green, Rab35; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Enlarged images are shown underneath. (J) Statistical analysis of the number of migrasomes per 10 0 μm retraction fiber per cell. The original images were captured as in I. n=119 for NRK; n=138 for NRK+Rab35-WT; n=133 for NRK+Rab35-7Glu. Mean±s.e.m., unpaired t-test.

FIG. 3A-3F illustrate Rab35 promotes migrasome formation. (A) Live-cell confocal microscopy images of WT and Rab35-KO NRK-mCherry-TSPAN4 cells. Red, TSPAN4. Scale bar, 10 μm. (B) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in A. n=72 for WT; n=80 for Rab35 KO. Mean±s.e.m., unpaired t-test. (C) Statistical analysis of retraction fiber length per cell. The original images were captured as in A. n=72 for WT; n=80 for Rab35 KO. Mean±s.e.m., unpaired t-test. (D) Live-cell confocal microscopy images of NRK-TSPAN4-GFP cells and NRK-TSPAN4-GFP cells stably expressing mCherry-Rab35-WT, mCherry-Rab35-S22N or mCherry-Rab35-Q67L. Green, TSPAN4; red, Rab35; yellow, merge. Scale bar, 10 μm. (E) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in D. n=133 for NRK; n=114 for NRK+Rab35-WT; n=140 for NRK+Rab35-S22N; n=89 for NRK+Rab35-Q67L. Mean±s.e.m., unpaired t-test. (F) Statistical analysis of retraction fiber length per cell. The original images were captured as in D. n=133 for NRK; n=114 for NRK+Rab35-WT; n=136 for NRK+Rab35-S22N; n=89 for NRK+Rab35-Q67L. Mean±s.e.m., unpaired t-test.

FIG. 4A-4L illustrate Rab35 recruits integrin to migrasome formation sites. (A) Live-cell SIM images of WT or Rab35-KO NRK-TSPAN4-mCherry cells stably expressing ITGα5-GFP. Green, ITGα5; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (B) The boxed regions from A were quantified for the ITGα5-GFP fluorescence intensity. (C) The images from A were quantified for the percentage of cells with condensed or diffuse ITGα5-GFP distribution. (D) Schematic representation of ITGα5 showing the amino acid sequence of the cytoplasmic domain in WT and the 5 mutants (1-5A, 2-5A, 3-5A, 4-5A, 5-5A) . (E) Live-cell SIM images of NRK-TSPAN4-mCherry cells stably expressing ITGα5 WT or 1-5A. Green, ITGα5; red, TSPAN4; yellow, merge. Scale bar, 10 μm. The boxed regions are enlarged at the right. (F) The boxed regions from E were quantified for the ITGα5-GFP fluorescence intensity. (G) Diagram of the dcFCCS assay: GFP-Rab35 (green dots) , Cy5-ITGα5-cyto (blue dots with blue lines) and ITGα5-cyto (blue lines) diffuse freely through the confocal detection volume of 488 nm (blue zone) and 640 nm (red zone) lasers. Only complexes containing both GFP and Cy5 fluorescence signals can contribute to cross-correlation curves. (H) dcFCCS curves of 100 nM GFP-Rab35-WT mixed with 500 nM Cy5-ITGα5-cyto-WT (blue) ; 100 nM GFP-Rab35-Q67L mixed with 500 nM Cy5-ITGα5-cyto-WT (green) ; and 100 nM GFP-Rab35-Q67L mixed with Cy5-ITGα5-cyto-5A (red) . (I) Sequences of Tat peptides. The Tat peptide (control, top) was fused to the WT cytoplasmic domain of ITGα5 (Tat-ITGα5-cyto-WT, middle) and the 5A mutant (Tat-ITGα5-cyto-5A, bottom) . (J) Live-cell SIM images of NRK cells expressing TSPAN4-mCherry and ITGα5-GFP, and treated with 100 μM control (Tat) , WT (Tat-WT) or mutant (Tat-5A) peptide from I. Green, ITGα5; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (K) The boxed regions from J were quantified for the ITGα5-GFP fluorescence intensity. (L) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in J. n=95 for Tat treatment, n=102 for Tat-WT treatment, n=89 for Tat-5A treatment. Mean±s.e.m., unpaired t-test.

FIG. 5A-5K illustrate The PI (4, 5) P ₂-Rab35 axis regulates migrasome formation in physiologically relevant settings and is evolutionarily conserved (A) Live-cell images of BJ cells treated with DMSO (Ctrl) and 20 μM ISA2011B. Green, WGA. Scale bar, 10 μm. (B) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in a. n=59 for Ctrl treatment, n=50 for ISA2011B treatment. Mean±s.e.m., unpaired t-test. (C) Live-cell images of BJ cells treated with NC shRNA and shPIP5K1A. Green, WGA. Scale bar, 10 μm. (D) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in (C) . n=50 for NC, n=50 for shPIP5K1A. Mean±s.e.m., unpaired t-test. (E) Live-cell images of BJ cells treated with 100 μM control (Tat) , WT (Tat-WT) or mutant (Tat-5A) peptide. Green, WGA. Scale bar, 10 μm. (F) Statistical analysis of the number of migrasomes per 100 μm retraction fiber per cell. The original images were captured as in (E) . n=64 for Tat treatment, n=75 for Tat-WT treatment, n=69 for Tat-5A treatment. Mean±s.e.m., unpaired t-test. (G) Live-cell images of gastrulation-stage zebrafish embryos treated with DMSO (Ctrl) and 100 μM ISA2011B. Red, PH-mCherry. Scale bar, 10 μm. (H) Statistical analysis of the number of migrasomes per embryo. The original images were captured as in (G) . n=102 for Ctrl treatment, n=97 for ISA2011B treatment. Mean±s.e.m., unpaired t-test. (I) Live-cell images of gastrulation-stage zebrafish embryos following injection with 100 μM control (Tat) , WT (Tat-WT) or mutant (Tat-5A) peptide at the 8-cell stage. Red, PH-mCherry. Scale bar, 10 μm. (J) Statistical analysis of the number of migrasomes per embryo. The original images were captured as in (I) . n=50 for Tat-WT treatment, n=39 for Tat-5A treatment. Mean±s.e.m., unpaired t-test. (K) Model for the role of the PI (4, 5) P ₂-Rab35 axis in migrasome formation.

FIG. 6A-6D illustrate migration of cell. (A) Quantification of the migration speed of WT and PIP5K1A KO NRK cells. Mean±s.e.m., unpaired t-test. (B) Live-cell confocal microscopy images of NRK cells expressing PLCD3-GFP and TSPAN4-mCherry. Green, PLCD3; red, TSPAN4; yellow, merge. Scale bar, 10 μm. Boxed regions are enlarged at the right. (C) Live-cell images of WT NRK-TSPAN4-mCherry cells, PLCD3-KO NRK-TSPAN4-mCherry cells, and PLCD3-KO NRK-TSPAN4-mCherry cells with rescue by GFP-PLCD3. Green, PLCD3; red, TSPAN4; yellow, merge. Scale bar, 10 μm. (D) The images from C were quantified for the migrasome number per 100 μm fiber per cell. Mean±S. D. . ****P＜0.0001; NS, not significant.

FIG. 7 illustrates Live-cell confocal microsopy images of NRK cells expressing TSPAN4-GFP and mCherry-tagged PIP ₂-binding proteins. Green, TSPAN4; red, PIP ₂-binding proteins; yellow, merge. Scale bar, 10 μm. Inserts show enlarged migrasomes.

FIG. 8 illustrates Live-cell confocal microscopy images of NRK-TSPAN4-mCherry cells expressing ITGα5-GFP WT and cytosolic domain mutants. Green, ITGα5; red, TSPAN4; yellow, merge. Scale bar, 10 μm.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the term “antibody” generally refers to a polypeptide molecule capable of specifically recognizing and/or neutralizing a specific antigen. For example, the antibody can include an immunoglobulin composed of at one or more heavy (H) chains and/or one or more light (L) chains, and include any molecule including its antigen binding portion. The term “antibody" includes monoclonal antibodies, antibodies fragment or antibody derivatives, including but not limited to, human antibodies, humanized antibodies, chimeric antibodies, single-strand antibodies (e.g., scFv) , and antigen-binding fragments of antibodies (e.g., Fab, Fab’ , VHH and (Fab) 2 fragments) .

As used herein, the term “antigen-binding fragment” generally refers to one or more fragments of the antibody which serve to specifically bind to the antigen. The antigen binding function of the antibody may be implemented by the full-length fragment of the antibody. The antigen binding function of the antibody may also be implemented by the followings: a heavy chain comprising a fragment of Fv, ScFv, dsFv, VHH, Fab, Fab’ or F (ab’ ) ₂, or a light chain comprising a fragment of Fv, ScFv, dsFv, Fab, Fab’ or F (ab’ ) ₂. (1) Fab fragment, that is, a monovalent fragment comprising VL, VH, CL and CH domains; (2) F (ab’ ) 2 fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (3) an Fd fragment comprising VH and CH domains; (4) an Fv fragment comprising VL and VH domains in one arm of an antibody; (5) a dAb fragment comprising a VH domain (Ward et al., (1989) Nature 341: 544-546) ; (6) isolated complementary determining region (CDR) ; and (7) a combination of two or more isolated CDRs which are optionally linked by a linker. Moreover, a monovalent single-strand molecule Fv (scFv) formed by pairing of VL and VH may further be included (see Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85: 5879-5883) .

As used herein, the term “dominant-negative” generally refers to a mutant or variant protein, or the gene encoding the mutant or variant protein, that substantially prevents a corresponding protein having wild-type function from performing the wild-type function. For example, it may refer to a gene or gene variant thereof that encodes a gene product that antagonizes the gene product of a wildtype gene.

As used herein, the term "engineered" generally refers to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome, of a polypeptide, or of other components. The term "engineered" can refer to alterations, additions, and/or deletions of the genes, polypeptides or other components. The term "engineered cell" generally refers to a modified cell of human or non-human origin. For example, an engineered cell can refer to a cell with an added, deleted and/or altered gene, polypeptide or other components.

As used herein, the term “ex vivo method” generally refers to a method with substantially all steps performed outside of an organism (e.g., an animal or a human body) . For example, an ex vivo method may be performed in or on a tissue from an organism in an external environment with minimal alteration of natural conditions. Tissues may be removed in many ways, including in part, as whole organs, or as larger organ systems. For example, in an ex vivo method, the samples to be tested may have been extracted from the organism. For example, using living cells or tissue from the same organism may also be considered to be ex vivo.

As used herein, the term "functional fragment" generally refers to a fragment having a partial region of a full-length protein or nucleic acid, but retaining or partially retaining the biological activity or function of the full-length protein or nucleic acid.

As used herein, the term "functional variant" generally refers to a nucleic acid molecule, or a polypeptide having similar amino acid or nucleic acid sequences as the parent sequence and retain one or more properties of the parent sequence.

As used herein, the term “in vitro method” generally refers to a method performed with microorganisms, cells, or biological molecules outside their normal biological context. For example, an in vitro method may be performed in labware such as test tubes, flasks, Petri dishes, and microtiter plates. In vitro methods may be performed using components of an organism that have been isolated from their usual biological surroundings. For example, microorganisms or cells can be studied in culture media, and proteins can be examined in solutions.

As used herein, the term “in vivo method” generally refers to a method wherein the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, and plants, as opposed to a tissue extract or dead organism. For example, the in vivo method may be performed in a whole organism, rather than in isolated cells thereof.

As used herein, the term “knock down” generally refers to a measurable reduction in the expression of a target mRNA or the corresponding protein in a genetically modified cell or organism as compared to the expression of the target mRNA or the corresponding protein in a counterpart control cell or organism that does not contain the genetic modification to reduce expression. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., siRNA, shRNA, microRNA, antisense RNA, or other RNA-mediated inhibition techniques, to knock down a target polynucleotide sequence.

As used herein, the term “knock out” generally includes deleting all or a portion of the target polynucleotide sequence in a way that interferes with the function of the target polynucleotide sequence. For example, a knock-out can be achieved by altering a target polynucleotide sequence by inducing a deletion in the target polynucleotide sequence in a functional domain of the target polynucleotide sequence. Those skilled in the art will readily appreciate how to use various genetic approaches, e.g., CRISPR/Cas systems, ZFN, TALEN, TgAgo, to knock out a target polynucleotide sequence or a portion thereof based upon the details described herein.

As used herein, the term “migrasome” generally refers to a membrane-bound cellular structure derived from or generated by a migrating cell. The term “migrasome” encompasses an organelle (also known as “pomegranate-like structure” or PLS) attached to a retraction fiber generated by a migrating cell. In some cases, the term “migrasome” also refers to a vesicle (e.g., an extracellular vesicle) already detached from the cell generating it. In the present disclosure, the term “migrasome” also refers to a vesicle (e.g., an artificial vesicle) with similar functions and/or compositions as such a vesicle or organelle derived from, and/or generated by migrating cells.

As used herein, the term “migrasome mediated biological process” generally refers to a biological process mediated by the formation, movement, function, degradation, and/or disintegration of a migrasome.

As used herein, the terms “migrating cell” and “circulating cell” are used interchangeably, and generally refer to a cell moving from one location to another location. In some cases, a migrating cell is a cell whose relative position, space, and/or contour has changed or is changing with time. A circulating cell comprises a cell circulating in the body fluid (e.g., blood or lymph) of an organism.

As used herein, the term “pharmaceutically acceptable excipient” generally refers to any material, which is inert in the sense that it substantially does not have a therapeutic and/or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties.

As used herein, the term “retraction fiber” or “RF” generally refers to actin-rich fibers exposed as the cell margin retracts. For example, the retraction fiber may include tubular strands left behind a cell during cell migration. During migration, RF may be pulled out at the trailing edge of cells, and migrasomes may form on the tips or branch points of the RF.

As used herein, the term “tetraspanin” generally refers to a membrane protein, which is also known as the transmembrane 4 superfamily (TM4SF) protein, and may have four transmembrane alpha-helices and two extracellular domains. For example, the term “tetraspanin” may encompass various isoforms of the tetraspanin, as well as the naturally-occurring allelic and processed forms thereof.

As used herein, the term “Tetraspanin 4 (TSPAN4) ” generally refers to a TSPAN4 gene and/or a protein that is encoded by the TSPAN4 gene. For example, the NCBI Entrez Gene for TSPAN4 may be 7106. For example, the UniProtKB/Swiss-Prot number for Tetraspanin 4 may be O14817. For example, the term “Tetraspanin 4” may encompass various isoforms of the Tetraspanin 4, the naturally-occurring allelic and processed forms thereof. The term also encompasses TSPAN4 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term TSPAN4 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term TSPAN4 encompasses the TSPAN4 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used herein, the term “Tetraspanin 9 (TSPAN9) ” generally refers to a TSPAN9 gene and/or a protein that is encoded by the TSPAN9 gene. For example, the NCBI Entrez Gene for TSPAN9 may be 10867. For example, the UniProtKB/Swiss-Prot number for Tetraspanin 9 may be O75954. For example, the term “Tetraspanin 9” may encompass the isoforms of the Tetraspanin 9, the naturally-occurring allelic and processed forms thereof. The term also encompasses TSPAN9 or a fragment thereof coupled to, for example, a tag (e.g., a histidine tag) , mouse or human Fc, or a signal sequence. The term TSPAN9 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. The term TSPAN9 encompasses the TSPAN9 gene or protein from any species, such as human, or a non-human animal, e.g., dog, mouse, rat, pig, monkey (e.g., Rhesus monkey) , cow, cat, chicken, zebrafish, etc.

As used herein, the term “PIP ₂” generally refers to Phosphatidylinositol bisphosphate. For example, Phosphatidylinositol 4, 5-bisphosphate. For example, the term “PIP ₂” may encompass the isoforms of the PIP ₂, the naturally-occurring allelic and processed forms thereof. The term PIP ₂ comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.

As used herein, the term “PI4P” generally refers to Phosphatidylinositol phosphate. For example, Phosphatidylinositol-4-phosphate. For example, the term “PI4P” may encompass the isoforms of the PI4P, the naturally-occurring allelic and processed forms thereof. The term PI4P comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof.

As used herein, the term “PIP5K1” generally refers to Phosphatidylinositol-4-Phosphate 5-Kinase Type 1. For example, Phosphatidylinositol-4-Phosphate 5-Kinase Type 1 A. For example, the term “PIP5K1” may encompass the isoforms of the PIP5K1, the naturally-occurring allelic and processed forms thereof. The term PIP5K1 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. For example, the term “PIP5K1” may refer to Q99755 in UniProt.

As used herein, the term “PLCD3” generally refers to Phospholipase C Delta 3. For example, the term “PLCD3” may encompass the isoforms of the PLCD3, the naturally-occurring allelic and processed forms thereof. The term PLCD3 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. For example, the term “PLCD3” may refer to Q8N3E9 in UniProt.

As used herein, the term “Rab35” generally refers to Ras-Related Protein Rab-35. For example, the term “Rab35” may encompass the isoforms of the Rab35, the naturally-occurring allelic and processed forms thereof. The term Rab35 comprises functional variants and/or fragments thereof, it also comprises orthologue and homologs thereof. For example, the term “Rab35” may refer to Q15286 in UniProt.

As used herein, the term “integrin α5” generally refers to Integrin Subunit Alpha 5 or ITGa5. For example, the term “ITGa5” may encompass the isoforms of the ITGa5, the naturally-occurring allelic and processed forms thereof. The term ITGa5 may comprise functional variants and/or fragments thereof, it also may comprise orthologue and homologs thereof. For example, the term “ITGa5” may refer to P08648 in UniProt.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “acomposition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

Detailed description

Migrasomes are recently discovered organelles, which are formed on the ends or branch points of retraction fibers at the trailing edge of migrating cells. The application showed that recruitment of integrins to the site of migrasome formation is essential for migrasome biogenesis. The application found that prior to migrasome formation, PIP5K1A, a PI4P kinase which converts PI4P into PI (4, 5) P ₂, is recruited to migrasome formation sites. The recruitment of PIP5K1A results in generation of PI (4, 5) P ₂ at the migrasome formation site. Once accumulated, PI (4, 5) P ₂ recruits Rab35 to the migrasome formation site by interacting with the C-terminal polybasic cluster of Rab35. The application further demonstrated that active Rab35 promotes migrasome formation by recruiting and concentrating integrin α5 at migrasome formation sites, which is possibly mediated by the interaction between integrin α5 and Rab35. The application identifies the upstream signaling events which orchestrate migrasome biogenesis.

Migrasomes are vesicular organelles which form on retraction fibers at the trailing edge of migrating cells. Migrasomes have important physiological functions including organ morphogenesis, mitochondrial quality control and lateral transfer of protein and mRNA between cells. During migrasome formation, integrins are first targeted to the ends or branch points of retraction fibers to form integrin foci. These foci will later grow into migrasomes and are operationally defined as migrasome formation sites. Once the integrin foci are formed, tetraspanin-enriched microdomains start to assemble at the migrasome formation site, and eventually expand into migrasomes. How integrins are targeted to migrasome formation sites is currently unknown.

Organelle biogenesis is a highly orchestrated process. Phosphoinositides are lipid signaling molecules which play a central role in organelle biogenesis. For example, during autophagosome formation, phosphatidylinositol 3-monophosphate (PI3P) -enriched structures (named omegasomes) are first formed on the ER, which then serve as a platform to recruit proteins which are essential for autophagosome formation. It is currently unclear whether migrasome formation is a regulated process which involves signaling pathway (s) .

PI (4, 5) P ₂ is a multi-functional lipid which regulates a large array of sub-cellular processes. PI (4, 5) P ₂ is the most abundant phosphoinositide and it is mainly localized in the plasma membrane. It is commonly believed that the majority of cellular PI (4, 5) P ₂ is synthesized by PIP5Ks, which convert PI4P to PI (4, 5) P ₂. In many cases, PI (4, 5) P ₂ carries out its functions through interaction with its partner proteins. So far, multiple PI (4, 5) P ₂ interaction domains, including PH, ANTH, ENTH and FERM, have been identified.

The application demonstrated that the biogenesis of migrasomes is a highly regulated process in which PI (4, 5) P ₂ signaling plays the central role. The application found that prior to migrasome formation, PI (4, 5) P ₂ is synthesized de novo at migrasome formation sites by PI5K1A, and migrasome formation is blocked when PI (4, 5) P ₂ generation is inhibited. Screening identified Rab35 as a migrasome-localized PI (4, 5) P ₂-binding protein. Further study revealed that Rab35 is essential for migrasome formation. Mechanistically, Rab35 is recruited to migrasome formation sites via interaction with PI (4, 5) P ₂. Subsequently, through Rab35-integrin α5 interaction, Rab35 recruits integrin α5 to migrasome formation sites, which prepares the sites for tetraspanin-dependent expansion.

The application proposes a provisional model for the signaling events that regulate migrasome biogenesis. The application proposes that the recruitment of PIP5K1A and de novo synthesis of PI (4, 5) P ₂ on the migrasome formation site is likely the triggering signal for migrasome formation. Once PI (4, 5) P ₂ reaches the concentration threshold, active Rab35 is recruited to the migrasome formation site through its polybasic cluster. Rab35 then serves as an adaptor to recruit integrins to the migrasome formation site. The interaction between active Rab35 and integrin thus creates the necessary adhesion point for migrasome formation.

The biogenesis of organelles is generally tightly regulated by signaling pathways. In many cases, lipid kinases are at the heart of these signaling cascades, which couple metabolic, mechanical and other cues to initiate the biogenesis of a particular organelle. The application reveals the essential role of the PI (4, 5) P ₂-Rab35 axis in migrasome formation. Migrasomes can be added to a growing list of organelles whose biogenesis is controlled by phosphoinositide signaling. Moreover, The application demonstrates that migrasome formation is an active biogenesis process which is tightly regulated by a signaling pathway, rather than a membrane shedding process in which membrane fragments are passively lost from the trailing edge of migrating cells.

The application found that PIP5K1A is recruited to the site of migrasome formation prior to formation of migrasomes. At this point, it might demonstrate how PIP5K1A is recruited to that particular location. It is possible that the recruitment is determined by a specific lipid/protein composition at the migrasome formation site; it is also possible that biophysical properties, such as membrane curvature, may contribute to the preferential recruitment of PIP5K1A.

The application observed the rapid accumulation of PI (4, 5) P ₂ after recruitment of PIP5K1A, which suggests that at least a proportion of the PI (4, 5) P ₂ on migrasome formation sites is synthesized de novo by PIP5K1A located at those sites. The fact that the highly enriched PI (4, 5) P ₂ signal on migrasomes does not diffuse onto the retraction fibers suggests that migrasome formation sites may have unique properties which favor the retention of PI (4, 5) P ₂. The application shows that de novo synthesis plus PI (4, 5) P ₂ retention may explain the rapid accumulation of PI (4, 5) P ₂ on migrasome formation sites.

The application shows that the targeting of integrin 5 to migrasome formation sites is dependent on active Rab35. The dual-color fluorescence cross-correlation spectroscopy analysis suggests that the cytosolic portion of integrin 5 can interact via its GFFKR motif with active Rab35. These data suggest that Rab35 may recruit integrin 5 to the migrasome formation site via direct interaction. It is worth noting that it might be detected as the Rab35/integrin 5 interaction by co-immunoprecipitation/western blotting. The application shows that this stems from the technical difficulty in detecting membrane protein interactions by immunoprecipitation. It remains possible that the interaction between Rab35 and integrin 5 may be an indirect one.

In one aspect, the present disclosure provides a method for regulating migrasome formation by a cell. The method may comprise regulating the amount and/or function of PIP _2.

In one aspect, the present disclosure provides an agent is for use in regulating migrasome formation and/or in regulating a migrasome-mediated biological process. The agent is capable of regulating the amount and/or function of PIP _2.

In one aspect, the present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell. The engineered cell has been modified to alter the amount and/or function of PIP ₂

In another aspect, the present disclosure provides use of the agent of the present disclosure and/or the engineered cell of the present disclosure in the preparation of a regulator for: i) migrasome formation; and/or ii) a migrasome-mediated biological process.

In one aspect, the present disclosure provides a method for regulating migrasome formation by a cell. The method may comprise regulating the amount and/or function of PIP5K1 in said cell.

In one aspect, the present disclosure provides an agent is for use in regulating migrasome formation and/or in regulating a migrasome-mediated biological process. The agent is capable of regulating the amount and/or function of PIP5K1 in said cell.

In one aspect, the present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell. The engineered cell has been modified to alter the amount and/or function of PIP5K1 in said cell.

In one aspect, the present disclosure provides a method for regulating migrasome formation by a cell. The method may comprise regulating the amount and/or function of Rab35 in said cell.

In one aspect, the present disclosure provides a method for regulating migrasome formation by a cell. The method may comprise regulating the amount and/or function of PI (4, 5) P ₂-binding molecule in said cell. For example, PI (4, 5) P ₂-binding molecule may comprise Rab35, Sdcbp, Twf1, Phlda3, Chmp3, Plcb1, Plcd1, Pard3, Anxa8, Svil, and/or Twf2.

In one aspect, the present disclosure provides an agent is for use in regulating migrasome formation and/or in regulating a migrasome-mediated biological process. The agent is capable of regulating the amount and/or function of Rab35 in said cell.

In one aspect, the present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell. The engineered cell has been modified to alter the amount and/or function of Rab35 in said cell.

In one aspect, the present disclosure provides a method for regulating migrasome formation by a cell. The method may comprise regulating the amount and/or function of integrin α5 (ITGa5) in said cell.

In one aspect, the present disclosure provides an agent is for use in regulating migrasome formation and/or in regulating a migrasome-mediated biological process. The agent is capable of regulating the amount and/or function of integrin α5 (ITGa5) in said cell.

In one aspect, the present disclosure provides an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell. The engineered cell has been modified to alter the amount and/or function of integrin α5 (ITGa5) in said cell.

In another aspect, the present disclosure provides a composition. The composition may comprise the agent according to the present disclosure, and/or the engineered cell according to the present disclosure.

In another aspect, the present disclosure provides a kit. The kit may comprise the agent according to the present disclosure, the engineered cell according to the present disclosure, and/or the composition according to the present disclosure.

According to any aspect of the present disclosure, migrasome formation may be promoted or inhibited.

For example, migrasome formation may be promoted by increasing the amount and/or function of PIP ₂ (including a functional derivative, a variant and/or a fragment thereof) . As another example, migrasome formation may be inhibited or decreased by decreasing or inhibiting the amount and/or function of PIP ₂ (including a functional derivative, a variant and/or a fragment thereof) .

For example, migrasome formation may be promoted by increasing the amount and/or function of PIP5K1 (including a functional derivative, a variant and/or a fragment thereof) . As another example, migrasome formation may be inhibited or decreased by decreasing or inhibiting the amount and/or function of PIP5K1 (including a functional derivative, a variant and/or a fragment thereof) .

For example, migrasome formation may be promoted by increasing the amount and/or function of Rab35 (including a functional derivative, a variant and/or a fragment thereof) . As another example, migrasome formation may be inhibited or decreased by decreasing or inhibiting the amount and/or function of Rab35 (including a functional derivative, a variant and/or a fragment thereof) .

For example, migrasome formation may be promoted by increasing the amount and/or function of cytosolic domain of ITGa5 (including a functional derivative, a variant and/or a fragment thereof) . As another example, migrasome formation may be inhibited or decreased by decreasing or inhibiting the amount and/or function of cytosolic domain of ITGa5 (including a functional derivative, a variant and/or a fragment thereof) .

Migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by observation, e.g. using microscopy, such as scanning electron microscope (SEM) and/or transmission electron microscope (TEM) . For example, migrasomes may be identified as membrane-bound vesicular structures, either in the extracellular space or in the cell generating them. The migrasomes may be connected to or closely associated with retraction fibers. A migrasome may be oval shaped, with diameters from e.g. about 400 nm to about 3500 nm, the migrasomes may contain multiple smaller vesicles. For example, the structure of a migrasome may resemble opened pomegranates (e.g., also known as pomegranate-like structures, or PLS) .

In addition or alternatively, migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by detecting the expression and/or amount of a migrasome specific marker. Such detection may be at transcriptional level and/or at protein level. Such marker may include but not limited to Tetraspanin-4, integrin, pleckstrin homology (PH) domain, NDST1 (bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1) , PIGK (phosphatidylinositol glycan anchor biosynthesis, class K) , CPQ (carboxypeptidase Q) and/or EOGT (EGF domain-specific O-linked N-acetylglucosamine transferase) .

In some cases, migrasome formation or a change thereof (e.g., an increase in migrasome formation or a decrease in migrasome formation) may be monitored and/or determined by staining the cell or sample with a migrasome specific dye, for example, by using WGA (wheatgerm agglutinin, a sialic acid-and N-acetyl-D glucosamine-binding lectin) .

In the present disclosure, promoting a migrasome-mediated biological process refers to causing a change in the biological process due to an increase of the amount and/or function of the migrasomes mediating such a biological process. In the present disclosure, inhibiting or decreasing a migrasome-mediated biological process refers to causing a change in the biological process due to a decrease of the amount and/or function of the migrasomes mediating such a biological process.

The migrasome-mediated biological process may be any process that could be affected by a migrasome. For example, such a biological process may involve migrasomes acting as packets of information which can be delivered to a spatially defined location to signal to the surrounding cells. In another example, the biological process may involve migrasomes acting as a garbage disposal mechanism by which damaged organelles are evicted from cells. In a further example, the biological process may involve migrasomes acting to mediate the lateral or horizontal transfer of RNAs and proteins. For example, such a biological process may be as reviewed by Yu and Yu, 2021 (doi: 10.1111/febs. 16183) . In some cases, the migrasome-mediated biological process is an in vitro process. In some cases, the migrasome-mediated biological process is an in vivo process. In some cases, the migrasome-mediated biological process is an ex vivo process. In some cases, the migrasome-mediated biological process may comprise cell-cell interactions (e.g., in cell cultures) .

The migrasome-mediated biological process may also include diseases and disorders, as reviewed by Yu and Yu, 2021 (doi: 10.1111/febs. 16183) . Such diseases and disorders may involve migrating cells, such as tumor metastasis, immune disorders, and developmental disorders. In some cases, such a disease or disorder may relate to brain tissue or cells, e.g., stroke. In some cases, such a disease or disorder may relate to kidney cells, such as kidney podocytes.

According to any aspect of the present disclosure, regulating the amount and/or function of Rab35 may comprise regulating the amount and/or function of the Rab35 on the plasma membrane of the cell. Regulating the amount and/or function of Rab35 may comprise increasing or decreasing the amount and/or function of the Rab35. For example, the amount of the Rab35 may be increased, while the function of the Rab35 may be increased, substantially unchanged or decreased. In some cases, the amount of the Rab35 is decreased, while the function of the Rab35 may be increased, substantially unchanged or decreased. In some cases, the function of the Rab35 is increased, while the amount of the Rab35 may be increased, substantially unchanged, or decreased. In some cases, the function of the Rab35 is decreased or inhibited, while the amount of the Rab35 may be increased, substantially unchanged, or decreased.

According to any aspect of the present disclosure, regulating the amount and/or function of PIP5K1 may comprise regulating the amount and/or function of the PIP5K1 on the plasma membrane of the cell. Regulating the amount and/or function of PIP5K1 may comprise increasing or decreasing the amount and/or function of the PIP5K1. For example, the amount of the PIP5K1 may be increased, while the function of the PIP5K1 may be increased, substantially unchanged or decreased. In some cases, the amount of the PIP5K1 is decreased, while the function of the PIP5K1 may be increased, substantially unchanged or decreased. In some cases, the function of the PIP5K1 is increased, while the amount of the PIP5K1 may be increased, substantially unchanged, or decreased. In some cases, the function of the PIP5K1 is decreased or inhibited, while the amount of the PIP5K1 may be increased, substantially unchanged, or decreased.

According to any aspect of the present disclosure, regulating the amount and/or function of cytosolic domain of ITGa5 may comprise regulating the amount and/or function of the cytosolic domain of ITGa5 on the plasma membrane of the cell. Regulating the amount and/or function of cytosolic domain of ITGa5 may comprise increasing or decreasing the amount and/or function of the cytosolic domain of ITGa5. for example, the amount of the cytosolic domain of ITGa5 may be increased, while the function of the cytosolic domain of ITGa5 may be increased, substantially unchanged or decreased. In some cases, the amount of the cytosolic domain of ITGa5 is decreased, while the function of the cytosolic domain of ITGa5 may be increased, substantially unchanged or decreased. In some cases, the function of the cytosolic domain of ITGa5 is increased, while the amount of the cytosolic domain of ITGa5 may be increased, substantially unchanged, or decreased. In some cases, the function of the cytosolic domain of ITGa5 is decreased or inhibited, while the amount of the cytosolic domain of ITGa5 may be increased, substantially unchanged, or decreased.

For example, regulating the amount and/or function of PIP ₂ on the plasma membrane of said cell. For example, said PIP ₂ may comprise PI (4, 5) P ₂. For example, said function of PIP ₂ may comprise recruiting Rab35 to formation site of said migrasome. For example, promoting said migrasome formation and/or promoting said migrasome-mediated biological process by increasing said amount and/or function of PIP ₂. For example, inhibiting said migrasome formation and/or inhibiting said migrasome-mediated biological process by decreasing said amount and/or function of PIP ₂.

For example, may comprise regulating the conversion of PI4P to PIP ₂. For example, regulating the amount and/or function of PIP ₂ may comprise regulating PI4P kinase. For example, regulating the amount and/or function of PIP ₂ may comprise regulating the expression and/or function of PIP5K1, the functional fragment thereof, and/or the functional variant thereof. For example, said PIP5K1 may comprise PIP5K1 alpha and/or PIP5K1 gamma.

For example, providing and/or overexpressing said PIP5K1 in said cell. For example, knocking out or knocking down the expression of a gene encoding for said PIP5K1 in said cell. For example, administering a PIP5K1 inhibitor to said cell. For example, said PIP5K1 inhibitor is a PIP5K1 selective inhibitor. For example, wherein said PIP5K1 inhibitor is ISA2011B, a dominant-negative PIP5K1, and/or derivatives thereof. For example, wherein said dominant-negative PIP5K1 may comprise a PIP5K1 L199 mutant and/or a PIP5K1 L207 mutant. For example, wherein said dominant-negative PIP5K1 may comprise a PIP5K1 L199I mutant and/or a PIP5K1 L207I mutant.

For example, regulating the degradation of said PIP ₂ into PI4P in said cell. For example, regulating the expression and/or function of PLCD3 in said cell. For example, the expression of a gene encoding for said PLCD3 has been knocked out or knocked down. For example, overexpressing a PLCD3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.

For example, providing and/or overexpressing said Rab35 in said cell. For example, providing and/or overexpressing a constitutively active form of Rab35 in said cell. For example, wherein said constitutively active form of Rab35 is Rab35 Q67 mutant. For example, wherein said constitutively active form of Rab35 is Rab35 Q67L mutant. For example, knocking out or knocking down the expression of a gene encoding for said Rab35 in said cell. For example, administering a Rab35 inhibitor to said cell. For example, wherein said Rab35 inhibitor is a Rab35 selective inhibitor. For example, wherein said Rab35 inhibitor is a Rab35 antibody, a dominant-negative Rab35, and/or derivatives thereof. For example, wherein said dominant-negative Rab35 may comprise a Rab35 S22 mutant. For example, wherein said dominant-negative Rab35 may comprise a Rab35 S22N mutant. For example, wherein said dominant-negative Rab35 may comprise a Rab35 mutant without positively charged residue. For example, wherein said dominant-negative Rab35 may comprise a Rab35 mutant with negatively charged Glutamic acid residue.

For example, providing and/or overexpressing said ITGa5 in said cell. For example, providing and/or overexpressing a cytosolic domain of ITGa5 in said cell. For example, providing and/or overexpressing GFFKR motif of cytosolic domain of ITGa5 in said cell. For example, providing and/or overexpressing ITGa5 and Rab35 binding protein in said cell. For example, knocking out or knocking down the expression of a gene encoding for said ITGa5 in said cell. For example, providing and/or overexpressing ITGa5 with no Rab35 binding in said cell. For example, administering a ITGa5 inhibitor to said cell. For example, said ITGa5 inhibitor is a ITGa5 selective inhibitor. For example, said ITGa5 inhibitor is a ITGa5 antibody, a dominant-negative ITGa5, and/or derivatives thereof. For example, said dominant-negative ITGa5 may comprise a ITGa5 mutant without cytosolic domain. For example, wherein said dominant-negative ITGa5 may comprise a ITGa5 mutant without GFFKR motif. For example, wherein said dominant-negative ITGa5 may comprise a ITGa5 mutant in which GFFKR is mutated. For example, wherein said dominant-negative ITGa5 may comprise a ITGa5 mutant in which GFFKR is mutated to AAAAA.

Knockouts may be accomplished through a variety of techniques. In some cases, the knockouts may be naturally occurring mutations that are screened out or identified (e.g., by DNA sequencing or other methods) .

In some cases, the knockouts are generated by homologous recombination. For example, it may involve creating a nucleic acid (e.g., DNA) construct containing the desired mutation. The construct may also comprise a drug resistance marker in place of the desired knockout gene. The construct may further contain a minimum length (e.g., 2kb or above) of homology to the target sequence. The construct may be delivered to target cells (for example, through microinjection, electroporation or other methods, such as transfection, using a virus or a non-virus system) . This method then relies on the cell’s own repair mechanisms to recombine the nucleic acid construct into the existing DNA (e.g., the genome of the cell) . This may result in the sequence of the gene being altered, and most cases the gene will be translated into a nonfunctional protein, if it is translated at all. The drug selection marker on the construct may be used to select for cells in which the recombination event has occurred. In diploid organisms, which contain two alleles for most genes, and may as well contain several related genes that collaborate in the same role, additional rounds of transformation and selection may be performed until every targeted gene is knocked out. Selective breeding may be required to produce homozygous knockout animals.

In some cases, the knockouts are generated using site-specific nucleases. Various methods may be used to precisely target a DNA sequence in order to introduce a double-stranded break. Once this occurs, the cell’s repair mechanisms will attempt to repair this double stranded break, often through non-homologous end joining (NHEJ) , which involves directly ligating the two cut ends together. This may be done imperfectly, therefore sometimes causing insertions or deletions of base pairs, which cause frameshift mutations. These mutations can render the gene in which they occur nonfunctional, thus creating a knockout of that gene.

For example, a zinc-finger nuclease may be used to generate such knockouts. Zinc-finger nucleases comprise DNA binding domains that can precisely target a DNA sequence. Each zinc finger can recognize codons of a desired DNA sequence, and therefore can be modularly assembled to bind to a particular sequence. These binding domains are coupled with a restriction endonuclease that can cause a double stranded break (DSB) in the DNA. Repair processes may introduce mutations that destroy functionality of the gene.

As another example, Transcription activator-like effector nucleases (TALENs) may be used to generate such knockouts. TALENs contain a DNA binding domain and a nuclease that can cleave DNA. The DNA binding region may comprise amino acid repeats that each recognize a single base pair of the desired targeted DNA sequence. If this cleavage is targeted to a gene coding region, and NHEJ-mediated repair introduces insertions and deletions, a frameshift mutation often results, thus disrupting function of the gene.

As a further example, clustered regularly interspaced short palindromic repeats (CRISPR) system may be used to generate such knockouts. The CRISPR/Cas9 method is a method for genome editing that contains a guide RNA complexed with a Cas9 protein. The guide RNA can be engineered to match a desired DNA sequence through simple complementary base pairing. The coupled Cas9 may cause a double stranded break in the DNA. Following the same principle as zinc-fingers and TALENs, the attempts to repair these double stranded breaks often result in frameshift mutations that result in a nonfunctional gene.

The knockout may also comprise a conditional gene knockout. A conditional gene knockout allows gene deletion in a tissue or cell when certain conditions are fulfilled, for example, in a tissue specific manner. It may be achieved by introducing short sequences called loxP sites around the gene. These sequences will be introduced into the germ-line via the same mechanism as a knock-out. This germ-line can then be crossed to another germline containing Cre-recombinase which is a viral enzyme that can recognize these sequences, recombines them and deletes the gene flanked by these sites.

Knocking down the CERT refers to a process by which the expression of the CERT encoding gene is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.

The knocking down may be through a genetic modification or may be transient. If a DNA of an organism or cell is genetically modified, the resulting organism or cell may be referred to as a “knockdown organism” or a “knockdown cell” . If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, and the result may be referred to as a “transient knockdown” .

In a transient knockdown, the binding of this oligonucleotide to the active gene or its transcripts causes decreased expression through a variety of processes. Binding can occur either through the blocking of transcription (in the case of gene-binding) , the degradation of the mRNA transcript (e.g. by small interfering RNA (siRNA) ) or RNase-H dependent antisense, or through the blocking of either mRNA translation, pre-mRNA splicing sites, or nuclease cleavage sites used for maturation of other functional RNAs, including miRNA (e.g. by morpholino oligos or other RNase-H independent antisense) .

RNA interference (RNAi) is a means of silencing genes by way of mRNA degradation. Gene knockdown by this method is achieved by introducing small double-stranded interfering RNAs (siRNA) into the cytoplasm. Small interfering RNAs can originate from inside the cell or can be exogenously introduced into the cell. Once introduced into the cell, exogenous siRNAs are processed by the RNA-induced silencing complex (RISC) . The siRNA is complementary to the target mRNA to be silenced, and the RISC uses the siRNA as a template for locating the target mRNA. After the RISC localizes to the target mRNA, the RNA is cleaved by a ribonuclease.

A “corresponding unmodified cell” refers to a cell that has not been modified to alter the amount and/or function of the target of interest therein, while with all the other features substantially the same as the engineered cell. In some cases, the corresponding unmodified cell is a wildtype cell (e.g., of the same cell type as the engineered cell) . In some cases, the corresponding unmodified cell may comprise one or more modifications, but the modification may be for other purposes.

The cell may be modified by any approach applicable for the purpose of the present disclosure. For example, the modification may be a genetic modification. In some cases, the modification may comprise treating the cell with one or more agent causing the desired change or effect. The modification may be temporary, transient or may be stable or permanent. In some cases, the engineered cell may be a progeny of a parent cell that has been modified.

The engineered cell may have an increased or decreased ability for forming migrasomes due to said modification.

In some cases, the engineered cell is a migrating cell or a circulating cell. In some cases, the engineered cell is seldomly migrating prior to such modification.

In the present disclosure, the engineered cell may be of any cell type. In some cases, the cell is a naturally present cell. In some cases, the cell is an artificially created or generated cell or a human made structure with cell-like characteristics. In some cases, the engineered cell may comprise a fibroblast cell (e.g., a L929 cell) . In some cases, the engineered cell may comprise an epithelial cell (such as an NRK cell) .

The present disclosure also provides use of the agent according to the present disclosure in the preparation of an engineered cell of the present disclosure.

In another aspect, the present disclosure also provides a method for generating an engineered cell of the present disclosure (e.g., an engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell) . The method may comprise altering the amount and/or function of PIP5K1 in the cell, as described in the present disclosure.

In the present disclosure, an agent may be a small molecule compound, an antibody, a nucleic acid molecule, a polypeptide, or fragments thereof. In some cases, the agent may comprise one or more active components, present in a single molecule or as separate molecules.

The agent may be provided in a non-active form and be converted into an active form in vitro or in vivo before, during or after administration.

The agent may be a pharmaceutical agent or an agent for non-pharmaceutical use.

The agent may exert the desired functions directly or indirectly via the function of additional agents, compositions or cells.

The composition of the present disclosure may be a pharmaceutical composition. The pharmaceutical composition may comprise a pharmaceutically acceptable excipient.

The composition may comprise an effective amount of the agent of the present disclosure. The effective amount may be an amount of the agent that when administered alone or in combination with another agent to a cell, tissue, or subject is effective to achieve the desired effect (e.g., regulating migrasome formation and/or in regulating a migrasome-mediated biological process) .

The compositions may further include pharmaceutically acceptable materials, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, i.e., carriers. These carriers are involved in transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

The formulation and delivery methods will generally be adapted according to the site and the disease to be treated. Exemplary formulations include, but are not limited to, those suitable for parenteral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration The dosage of the agents of the disclosure will vary according to the extent and severity of the need for regulation, the activity of the administered composition, the general health of the subject, and other considerations well known to the skilled artisan.

Agents as described herein can be incorporated into compositions suitable for administration. Such compositions typically comprise the agent and a pharmaceutically acceptable carrier. Supplementary active compounds can also be incorporated into the compositions. In yet other embodiments, the agents described herein are delivered locally. Localized delivery allows for the delivery of the agent non-systemically, for example, to the site of regulation in need.

The kit of the present disclosure may comprise the agent, the engineered cell, and/or the composition according to the present disclosure.

The agent, the engineered cell, and/or the composition may be comprised in suitable packaging, and written material that can include instructions for use, discussion of experimental studies (such as clinical studies) , listing of side effects, and the like. Such kits may also include information, such as scientific literature references, package insert materials, experimental results (such as clinical trial results) , and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the agent, the engineered cell and/or the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the users (such as health care provider or consumers) . Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.

The kit may further contain an additional agent. In some embodiments, the agent, engineered cell and/or the composition of the present invention and the additional agent may be provided as separate compositions in separate containers within the kit. In some embodiments, the agent, the engineered cell and/or the composition of the present disclosure and the additional agent are provided as a single composition within a container in the kit. Suitable packaging and additional articles for use (e.g., measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like) are known in the art and may be included in the kit. Kits described herein can be provided, marketed and/or promoted to users (such as health providers) , including scientists, physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Experimental model and subject details

NRK, MGC803, BJ cells and their derivatives were cultured at 37℃ and 5%CO ₂ in DMEM medium supplemented by 10%serum, 1%Glutamax and 1%penicillin–streptomycin.

For NRK transfection, one third of a full 6-cm dish of cells was transfected with 3-5 μg plasmid via Amaxa nucleofection using solution T and program NRK. For transfection of MGC803 cells, a 3.5-cm dish of 70-90%confluent cultured cells was transfected with 5 μg DNA via a Lipofectamine-3000 transfection kit (Invitrogen) .

All wild-type (WT) zebrafish used in this study were from the Tuebingen (Tu) strain.

Constructs

For all the PIP ₂-binding proteins identified in the MS screen, the corresponding genes were cloned from rat cDNA and transferred into pmCherry-C1 and N1. GFP-PIP5K1A, GFP-PLCD3, GFP-Rab35 and their derivatives were cloned in pB-GAG-BGH. PLCγ-PH-GFP was cloned in pEGFP-N1. ITGα5. ITGα5 mutations were generated from original plasmid. His-GFP-Rab35 and His-GFP-Rab35-Q67L were cloned in pET21b.

Generation of KO cell lines

To generate knockout cell lines, the PIP5K1A, PLCD3 and Rab35 genes in NRK cells were deleted by a modified PX458 plasmid. The sgRNA sequences were:

PIP5K1A-gRNA-1-F 5’ -CACCGGATAAACAGGCAGTGGCTG-3’ (SEQ ID NO: 1)

PIP5K1A-gRNA-1-R 5’ -AAACCAGCCACTGCCTGTTTATCC-3’ (SEQ ID NO: 2)

PIP5K1A-gRNA-2-F 5’ -CACCGAGTTGGTGGAGGCTAAGGG-3’ (SEQ ID NO: 3)

PIP5K1A-gRNA-2-R 5’ -AAACCCCTTAGCCTCCACCAACTC-3’ (SEQ ID NO: 4)

PLCD3-gRNA-1-F5’ -CACCGTTCGCCCCTGCTAGTGAGT-3’ (SEQ ID NO: 5)

PLCD3-gRNA-1-R 5’ -AAACACTCACTAGCAGGGGCGAAC-3’ (SEQ ID NO: 6)

PLCD3-gRNA-2-F5’ -CACCGCACCAAAAGGCCCGGGCTA-3’ (SEQ ID NO: 7)

PLCD3-gRNA-2-R 5’ -AAACTAGCCCGGGCCTTTTGGTGC-3’ (SEQ ID NO: 8)

Rab35-gRNA-1-F 5’ -CACCGGCGACCAGGGTGCACCCCA-3’ (SEQ ID NO: 9)

Rab35-gRNA-1-R 5’ -AAACTGGGGTGCACCCTGGTCGCC-3’ (SEQ ID NO: 10)

Rab35-gRNA-2-F 5’ -CACCGGAGGCGGTGCGGGCCCTGC-3’ (SEQ ID NO: 11)

Rab35-gRNA-2-R 5’ -AAACGCAGGGCCCGCACCGCCTCC-3’ (SEQ ID NO: 12)

After 48 h transfection, cells were sorted for GFP signal by fluorescence-activated cell sorting (FACS) and seeded into 96-well plates. For PIP5K1A and Rab35, KO clones were identified by western blotting. For PLCD3, KO clones were identified by PCR, which yielded a smaller product. Primers for PLCD3 knockout identification were:

ID-PLCD3-F: 5’ -GTCAGAATTCCCAGAAAAAAGTGTCTGC-3’ (SEQ ID NO: 13)

ID-PLCD3-R: 5’ -GAAGCCAGTTAGCCCGTACACC-3’ (SEQ ID NO: 14)

Immunofluorescence

Cells were washed with phosphate buffered saline (PBS) , then fixed in medium: 4%paraformaldehyde =1: 1 for 5 min, followed by 4%paraformaldehyde for 5 min. Fixed cells were permeabilized and blocked in 0.05%saponin, 10%FBS in PBS for 30 min, stained with antibody according to the manufacturer’s instructions with 10%FBS in PBS at 4℃ overnight, and washed with PBS three times. Cells were stained with secondary antibody with 10%FBS in PBS for 1 h and washed with PBS three times.

Imaging and image analysis

Confocal snapshot images were acquired using a Fluoview 1000 confocal microscope (Olympus) , and a NIKON A1. Images were collected at 1,024×1,024 pixels. Long-term time-lapse images of living cells were collected using a NIKON A1 microscope. Images were collected at 1,024×1,024 pixels. SIM snapshot images were collected by SIM set up on the NIKON A1. Fluorescence intensities of snapshot images were analysed using ImageJ Fiji, and statistical analyses were conducted using Graphpad Prism 7. Fluorescence intensities of long-term time-lapse images were statistically analysed by NIS-element analysis software.

To acquire two-dimensional images and living two-dimensional images of embryos, mRNA was injected at the desired embryonic stage. The embryos were then embedded in 1%low-melting-point agarose and imaged by Dragonfly spinning disk microscopy.

Protein purification

pET21b-His-GFP-Rab35 was expressed in E. coli BL21 (DE3) cells cultured at 16℃ for 18 h with induction by isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM. His-GFP-Rab35 was purified by Ni ²⁺-NTA agarose affinity chromatography in 20 mM HEPES 8.0, 100 mM NaCl, 2 mM MgCl ₂, 1 mM DTT and cocktail buffer.

Peptide synthesis

The sequences of peptides from the integrin α5 cytoplasmic region were

WT: YKLGFFKRSLPYGTAMEKAQLKPPATSDA (SEQ ID NO: 15)

5A: YKLAAAAASLPYGTAMEKAQLKPPATSDA (SEQ ID NO: 16)

Tat-WT: YGRKKRRQRRRGGYKLGFFKRSLPYGTAMEKAQLKPPATSDA (SEQ ID NO: 17)

Tat-5A: YGRKKRRQRRRGGYKLAAAAASLPYGTAMEKAQLKPPATSDA (SEQ ID NO: 18)

Tat: YGRKKRRQRRRGG (SEQ ID NO: 19)

Peptides were synthesized accordingly.

ITGα5-cyto peptide labeling

Synthesized wild-type or mutant ITGα5-cyto peptide was mixed with Sulfo-Cyanine5 (Cy5) NHS ester (Lumiprobe) at 1: 1 molar ratio in reaction buffer (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl ₂) and incubated for 2 hours at 25 ℃. For the control, Tris pH8.0 was mixed with Sulfo-Cyanine5 (Cy5) NHS ester at 7: 1 molar ratio in reaction buffer, as ITGα5-cyto-WT peptide has 7 -NH ₂ groups. To fully quench the reaction, 20-fold Tris pH 8.0 was added to react with the excess free dye for 20 minutes and then the mixture was centrifuged at 13, 000 rpm for 10 min to remove the precipitate. The supernatant was used for the following dcFCCS assay.

dcFCCS measurements and data analysis

dcFCCS measurements were conducted on a home-built confocal microscope, based on a Zeiss AXIO Observer D1 fluorescence microscope equipped with solid-state 488 nm and 640 nm excitation lasers (Coherent Inc. OBIS Smart Lasers) , an oil-immersion objective (Zesis, 100×, numerical aperture = 1.4) and avalanche photodiode detectors (APDs, Excelitas, SPCM-AQRH-14) . Fluorescence passed through a pinhole (50 μm diameter) , then was split by a T635lpxr dichroic mirror (Chroma) . Bandpass filters ET525/50m (Chroma) and ET700/75m (Chroma) were used to further filter fluorescence for GFP and Cy5 detection channels, respectively.

Wild-type or mutated GFP-Rab35 protein was centrifuged at 13, 000 rpm for 10 min to remove aggregates. dcFCCS experiments were carried out with 488 nm and 640 nm laser excitation at 25 ℃. GFP-Rab35 and ITGα5-cyto were mixed in 20 mM HEPES pH 7.5, 100 mM NaCl, 2 mM MgCl ₂, 0.1%BSA and then loaded immediately onto coverslips passivated with polyethylene glycol. Raw data of photon arrival time was recorded for 30 min. Three repeats were performed for each experimental condition.

Zebrafish mRNA and peptide injection

For live-cell imaging, one cell of a zebrafish embryo at the eight-cell stage was injected with 100 pg PH-mCherry mRNA. For peptide injection, 1 nL of 1 mM Tat, Tat-WT or Tat-5A was injected into the yolk at the eight-cell stage.

Example 1

Generation of PI (4, 5) P ₂ by PIP5K1A at the migrasome formation sites

The application found that PLCγ-PH-GFP, a probe for PI (4, 5) P ₂, can label migrasomes, which the application confirmed here (Figure 1A) . Staining of cells using an anti-PI (4, 5) P ₂ antibody also showed enrichment of the PI (4, 5) P ₂ signal in migrasomes (Figure 1B) . These results suggest that migrasomes contain PIP ₂. To study the dynamics of PIP ₂ on migrasomes, the application carried out time-lapse imaging. The application found that PLCγ-PH-GFP was recruited to migrasome before TSPAN4 (Figure 1C) . The application reported that Prior to recruitment of TSPAN4, integrin α5 forms foci on retraction fibers which define the sites for migrasome formation. Next, the application checked the dynamics of PLCγ-PH-TagBFP compared to integrin 5. The application found that the recruitment of PLCγ-PH-TagBFP is slightly faster than integrin 5 (Figure 1D, E) . Together, these data suggest that PI (4, 5) P ₂ is generated on or recruited to the migrasome formation site prior to migrasome growth.

PI (4, 5) P ₂ can be generated by PI4P kinase, which converts PI4P into PI (4, 5) P ₂. To test whether PI4P kinase is involved in generation of PI (4, 5) P ₂ at the site of migrasome formation, the application stained cells with an antibody against PIP5K1A, the major isoform of PI4P kinase expressed in NRK cells. The application found that indeed PIP5K1A is localized on migrasomes (Figure 1F) . Similarly, ectopically expressed PIP5K1A-GFP is localized on migrasomes, and time-lapse imaging showed that PIP5K1A-GFP is recruited to the site of migrasome formation prior to the recruitment of TSPAN4 (Figure 1G) . This is consistent with the appearance of the PI (4, 5) P ₂ signal (Figure 1C) .

Next, the application tested whether PIP5K1A is responsible for PI (4, 5) P ₂ generation at the sites of migrasome formation. To test this idea, the application treated cells with ISA2011B, a PIP5K1A inhibitor. The application found that ISA2011B treatment blocked migrasome formation (Figure 1H, I) . To further confirm that PI (4, 5) P ₂ is required for migrasome formation, the application generated PIP5K1A knockout cells, and found that indeed formation of migrasomes is markedly reduced (Figure 1J, K) . Ectopically expressing wild-type PIP5K1A, but not two kinase-dead PIP5K1A mutants, restored migrasome formation (Figure 1J, K) . This suggests that the enzyme activity of PIP5K1A is required for migrasome formation. This provides further evidence that the level of PI (4, 5) P ₂ is a determinant of migrasome formation. It is worth noting that knockout of PIP5K1A does not affect retraction fiber formation or cell migration (Figure 6A) . Together, these results suggest that generation of PI (4, 5) P ₂ by PIP5K1A at the site of migrasome formation is required for migrasome biogenesis.

Since PI (4, 5) P ₂ can be hydrolyzed by lipid phosphatases, the application next checked the localization of the known PI (4, 5) P ₂ phosphatases. The application found that PLCD3 is localized on migrasomes (Figure 6B) . To further test the role of PI (4, 5) P ₂ in migrasome formation, the application generated a PLCD3 knockout cell line. In this cell line, the formation of migrasomes is significantly enhanced (Figure 6C, D) , and ectopic expression of PLCD3 returns migrasome formation to the normal level. Together, these data provide further evidence to support the role of PI (4, 5) P ₂ in migrasome formation.

Example 2

PIP ₂ recruits Rab35 to migrasome formation sites

Next, the application investigated how PI (4, 5) P ₂ regulates migrasome formation. It shows that PI (4, 5) P ₂ may regulate migrasome formation by recruiting PI (4, 5) P ₂-binding proteins which are required for migrasome formation. To screen the possible migrasome-localized PI (4, 5) P ₂-binding proteins, the application first compiled a list of all the PI (4, 5) P ₂-binding proteins in the rat genome. Next, the application compared this list to the list of proteins in migrasomes, which the application identified by mass spectrometry analysis of purified migrasomes. The application found 23 PI (4, 5) P ₂- binding proteins on the mass spectrometry list, including Rab35 (Figure 2A) . The application then generated mCherry-tagged constructs for 19 of these proteins, and found that some of them are localized on migrasomes (Figure 7) . Among these proteins, the application picked Rab35 for further study, since Rabs play key roles in organelle biogenesis.

The application first confirmed the recruitment of Rab35 by staining cells with anti-Rab35 antibody. The application found that endogenous Rab35 is indeed localized on migrasomes and on small puncta along the retraction fiber (Figure 2B) . Similarly, ectopically expressed mCherry-Rab35 is localized on migrasomes and on migrasome formation sites (Figure 2C) . To study the dynamics of Rab35 recruitment, the application carried out time-lapse imaging using Rab35-mCherry (Figure 2D) . The application found that the Rab35 signal is first evenly and diffusely distributed along a retraction fiber. Prior to migrasome formation, the Rab35 signals are gradually concentrated at the branch points and they become more intense. Eventually, the Rab35-positive puncta start to enlarge and grow into migrasomes (Figure 2D) . These results suggest that Rab35 is recruited to the sites of migrasome formation prior to migrasome biogenesis.

Next, the application tested whether the Rab35 recruitment is PI (4, 5) P ₂-dependent. The application treated cells with the PIP5K1A inhibitor ISA2011B, and observed impaired recruitment of Rab35 to migrasome formation sites (Figure 2E, F) . Similarly, Rab35 failed to be recruited to the sites of migrasome formation in PIP5K1A knockout cells (Figure 2G, H) . These results confirm that the recruitment of Rab35 is PI (4, 5) P ₂-dependent. It showed that PI (4, 5) P ₂ recruits Rab35 to the plasma membrane by interacting with its C-terminal polybasic amino acid cluster, which consists of a stretch of positively charged Lys and Arg residues. When the application replaced the polybasic cluster with negatively charged Glu (Rab35-7Glu-tail) , the application found that the mutant Rab35 cannot bind to the plasma membrane and fails to promote migrasome formation (Figure 2I, J) . Together, these data suggest that Rab35 is recruited to the sites of migrasome formation by PI (4, 5) P ₂.

Example 3

Rab35 is required for migrasome formation

Next, to check whether or not Rab35 is required for migrasome formation, the application generated a Rab35 knockout cell line. The application found that knockout of Rab35 severely impairs migrasome formation (Figure 3A, B) . Interestingly, knockout of Rab35 enhances the number and length of retraction fibers (Figure 3A, C) . To further confirm the role of Rab35 in migrasome formation, the application established three cell lines stably expressing wild type Rab35, a dominant negative Rab35 mutant (S22N) and a constitutively active form of Rab35 (Q67L) . The application found that overexpressing wild-type and constitutively active Rab35 enhanced migrasome formation, while expressing dominant negative Rab35 reduced migrasome formation (Figure 3D, E) . Moreover, expressing dominant negative Rab35 enhanced retraction fiber formation (Figure 3F) .

Example 4

Rab35 promote migrasome formation by targeting integrin α5 to migrasome

Finally, the application investigated how Rab35 promotes migrasome formation. It showed that Rab35 is required for integrin trafficking. This prompted us to investigate the relationship between integrin and Rab35. The application showed that integrin α5 (ITGα5-GFP) marks the migrasome formation sites and determines migrasome formation. In Rab35 KO cells, instead of concentrating at the migrasome formation sites, ITGα5-GFP is evenly and diffusely distributed along retraction fibers (Figure 4A-C) . This suggests that the targeting of integrin to migrasome formation sites is impaired.

Next, the application investigated the molecular mechanism underlying the Rab35-dependent recruitment of ITGα5-GFP. It shows that all integrin subunits can associate with Rab21 via the conserved membrane-proximal GFFKR motif, which is also present in integrin α5. It might be that whether or not Rab35 can associate with integrin α5 through this motif. To test this hypothesis, the application generated an integrin α5 mutant in which GFFKR is mutated to AAAAA (1-5A) . As controls, the application also generated another 4 mutants in which the 4 successive sets of 5 consecutive amino acids in the cytosolic portion of integrin α5 are mutated to AAAAA (2-5A, 3-5A, 4-5A, 5-5A) (Figure 4D) . Together, these mutants cover the majority of the integrin α5 cytosolic domain. The application found that the GFFKR/AAAAA (1-5A) mutant, but not any of the other mutants, showed impaired targeting to migrasome formation sites (Figure 4E, F) . This suggests that the GFFKR motif is required for targeting integrin α5 to migrasome formation sites, possibly by affecting the association with Rab35.

Next, the application directly tested the possible association between Rab35 and integrin α5. Due to the difficulty associated with reliably detecting interactions involving membrane proteins by immunoprecipitation, the application used dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to capture the interaction between Rab35 and the cytosolic domain of integrin α5 (ITGα5-cyto) (Figure 4G) . To perform the assay, the application first purified GFP-Rab35-WT and GFP-Rab35-Q67L (constitutively active mutant) proteins. The application also synthesized ITGα5-cyto-WT and ITGα5-cyto-1-5A labeled with the fluorophore Cyanine5 (Cy5) . When GFP-Rab35-WT was mixed with Cy5-ITGα5-cyto-WT or Cy5 fluorophore, neither of the mixtures exhibited significant cross-correlation signals (Figure 4H) , which indicates no binding between WT Rab35 and Cy5-ITGα5-cyto-WT. However, the constitutively active mutant GFP-Rab35-Q67L exhibited strong cross-correlation signals with Cy5-ITGα5-cyto-WT under similar experimental conditions (Figure 4H) , which indicates that GFP-Rab35-Q67L can bind to Cy5-ITGα5-cyto-WT. In contrast, GFP-Rab35-Q67L had no cross-correlation signals with Cy5-ITGα5-cyto-1-5A (Figure 4H) , which suggests that the GFFKR motif is required for the binding between the cytosolic domain of integrin α5 and the active form of Rab35. It is worth noting that recombinant GFP-Rab35-WT was purified from E. coli, and thus should be in the inactive form. These data suggest that active Rab35 can bind to the cytosolic domain of integrin α5 through the GFFKR motif.

The application reasoned that if Rab35 recruits integrin α5 to migrasome formation sites by interacting with the GFFKR motif of integrin α5, then loading cells with integrin α5-derived peptides containing the GFFKR motif should competitively inhibit the Rab35-mediated recruitment of integrin α5 to migrasome formation sites and reduce migrasome formation. Indeed, the application found that treating cells with the plasma membrane-permeable peptide ITGα5-cyto-WT reduced both the targeting of integrin to migrasome formation sites and migrasome formation; in contrast, treating cells with the GFFKR/AAAAA mutant peptide failed to block integrin targeting or migrasome formation (Figure 4I-L) . These results suggest that Rab35 promotes migrasome formation by targetting integrin to migrasome formation sites.

Example 5

The PI (4, 5) P ₂-Rab35 axis regulates migrasome formation in physiologically relevant settings and is evolutionarily conserved

Lastly, the application tested whether the PI (4, 5) P ₂-Rab35 axis regulates migrasome formation in diverse settings. The application first tested BJ cells, a fibroblast cell line established from skin taken from normal foreskin from a neonatal male. The application found that treating BJ cells with the PIP5K1A inhibitor ISA2011B (Figure 5A, 5B) , or knocking down PIP5K1A (Figure 5C, 5D) , significantly impaired migrasome formation. Moreover, treating BJ cells with ITGα5-cyto-WT, but not the GFFKR/AAAAA mutant peptide, blocked migrasome formation (Figure 5E, 5F) . These observations suggest that the regulation of migrasome formation by the PI (4, 5) P ₂-Rab35 axis is conserved in human cells. Finally, the application tested whether the PI (4, 5) P ₂-Rab35 axis regulates migrasome formation in vivo. The application that migrasomes are formed during zebrafish embryonic development. Here, the application found that treating zebrafish embryos with PIP5K1A inhibitor (Figure 5G, 5H) or ITGα5-cyto-WT peptide, but not the GFFKR/AAAAA mutant peptide (Figure 5I, 5J) , significantly reduced migrasome formation. Together, these findings suggest that the PI (4, 5) P ₂-Rab35 axis regulates migrasome formation in a range of physiological settings and is conserved in different vertebrates.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of PIP ₂ in said cell.
The method of claim 1, said method comprises regulating the amount and/or function of PIP ₂ on the plasma membrane of said cell.
The method of any one of claims 1-2, said PIP ₂ comprises PI (4, 5) P ₂.
The method of any one of claims 1-3, said function of PIP ₂ comprises recruiting Rab35 to formation site of said migrasome.
The method of any one of claims 1-4, said method comprises promoting said migrasome formation and/or promoting said migrasome-mediated biological process by increasing said amount and/or function of PIP ₂.
The method of any one of claims 1-5, said method comprises inhibiting said migrasome formation and/or inhibiting said migrasome-mediated biological process by decreasing said amount and/or function of PIP ₂.
The method of any one of claims 1-6, said regulating the amount and/or function of PIP ₂ comprises regulating the conversion of PI4P to PIP ₂.
The method of claim 7, said regulating the amount and/or function of PIP ₂ comprises regulating PI4P kinase.
The method of any one of claims 7-8, said regulating the amount and/or function of PIP ₂ comprises regulating the expression and/or function of PIP5K1, the functional fragment thereof, and/or the functional variant thereof.
The method of claim 9, said PIP5K1 comprises PIP5K1 alpha and/or PIP5K1 gamma.
The method of any one of claims 9-10, wherein said regulating the expression and/or function of PIP5K1 comprises providing and/or overexpressing said PIP5K1 in said cell.
The method of any one of claims 9-11, wherein said regulating the expression and/or function of PIP5K1 comprises knocking out or knocking down the expression of a gene encoding for said PIP5K1 in said cell.
The method of any one of claims 9-12, wherein said regulating the expression and/or function of PIP5K1 comprises administering a PIP5K1 inhibitor to said cell.
The method of claim 13, wherein said PIP5K1 inhibitor is a PIP5K1 selective inhibitor.
The method of any one of claims 13-14, wherein said PIP5K1 inhibitor is ISA2011B, a dominant-negative PIP5K1, and/or derivatives thereof.
The method of claim 15, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199 mutant and/or a PIP5K1 L207 mutant.
The method of any one of claims 15-16, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199I mutant and/or a PIP5K1 L207I mutant.
The method of any one of claims 1-17, wherein said regulating the amount and/or function of PIP ₂ comprises regulating the degradation of said PIP ₂ into PI4P in said cell.
The method of claim 18, wherein said regulating the amount and/or function of PIP ₂ comprises regulating the expression and/or function of PLCD3 in said cell.
The method of claim 19, wherein the expression of a gene encoding for said PLCD3 has been knocked out or knocked down.
The method of any one of claims 18-20, wherein said regulating the amount and/or function of PIP ₂ comprises overexpressing a PLCD3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
An engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of PIP ₂ therein.
The engineered cell of claim 22, said engineered cell has been modified to alter the amount and/or function of PIP ₂ on the plasma membrane of said cell.
The engineered cell of any one of claims 22-23, said PIP ₂ comprises PI (4, 5) P ₂.
The engineered cell of any one of claims 22-24, said function of PIP ₂ comprises recruiting Rab35 to formation site of said migrasome.
The engineered cell of any one of claims 22-25, said engineered cell has increased ability for forming migrasomes, and has been modified to increase said amount and/or function of PIP ₂ therein.
The engineered cell of any one of claims 22-25, said engineered cell has decreased ability for forming migrasomes, and has been modified to decrease said amount and/or function of PIP ₂ therein.
The engineered cell of any one of claims 22-27, said engineered cell has been modified to alter the conversion of PI4P to PIP ₂.
The engineered cell of claim 28, said engineered cell has been modified to alter PI4P kinase.
The engineered cell of any one of claims 28-29, said engineered cell has been modified to alter the expression and/or function of PIP5K1, the functional fragment thereof, and/or the functional variant thereof.
The engineered cell of claim 30, said PIP5K1 comprises PIP5K1 alpha and/or PIP5K1 gamma.
The engineered cell of any one of claims 30-31, said engineered cell has been modified to provide and/or overexpress said PIP5K1.
The engineered cell of claim 32, wherein the expression of a gene encoding for said PIP5K1 has been knocked out or knocked down.
The engineered cell of any one of claims 32-33, said engineered cell has been treated with a PIP5K1 inhibitor.
The engineered cell of claim 34, wherein said PIP5K1 inhibitor is a PIP5K1 selective inhibitor.
The engineered cell of any one of claims 34-35, wherein said PIP5K1 inhibitor is ISA2011B, a dominant-negative PIP5K1, and/or derivatives thereof.
The engineered cell of claim 36, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199 mutant and/or a PIP5K1 L207 mutant.
The engineered cell of any one of claims 36-37, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199I mutant and/or a PIP5K1 L207I mutant.
The engineered cell of any one of claims 22-38, said engineered cell has been modified to alter the degradation of said PIP ₂ into PI4P in said cell.
The engineered cell of claim 39, said engineered cell has been modified to alter the expression and/or function of PLCD3 in said cell.
The engineered cell of claim 40, wherein the expression of a gene encoding for said PLCD3 has been knocked out or knocked down.
The engineered cell of any one of claims 39-41, said engineered cell has been modified to overexpress a PLCD3, a functional fragment thereof, a functional variant thereof, and/or a nucleic acid molecule encoding one or more of them.
A method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of PIP5K1 in said cell.
The method of claim 43, said PIP5K1 comprises PIP5K1 alpha and/or PIP5K1 gamma, the functional fragment thereof, and/or the functional variant thereof.
The method of any one of claims 43-44, wherein said regulating the expression and/or function of PIP5K1 comprises providing and/or overexpressing said PIP5K1 in said cell.
The method of any one of claims 43-44, wherein said regulating the expression and/or function of PIP5K1 comprises knocking out or knocking down the expression of a gene encoding for said PIP5K1 in said cell.
The method of any one of claims 43-44, wherein said regulating the expression and/or function of PIP5K1 comprises administering a PIP5K1 inhibitor to said cell.
The method of claim 47, wherein said PIP5K1 inhibitor is a PIP5K1 selective inhibitor.
The method of any one of claims 47-48, wherein said PIP5K1 inhibitor is ISA2011B, a dominant-negative PIP5K1, and/or derivatives thereof.
The method of claim 49, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199 mutant and/or a PIP5K1 L207 mutant.
The method of any one of claims 49-50, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199I mutant and/or a PIP5K1 L207I mutant.
An engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of PIP5K1 therein.
The engineered cell of claim 52, said PIP5K1 comprises PIP5K1 alpha and/or PIP5K1 gamma.
The engineered cell of any one of claims 52-53, said engineered cell has been modified to provide and/or overexpress said PIP5K1.
The engineered cell of any one of claims 52-53, wherein the expression of a gene encoding for said PIP5K1 has been knocked out or knocked down.
The engineered cell of any one of claims 52-53, said engineered cell has been treated with a PIP5K1 inhibitor.
The engineered cell of claim 56, wherein said PIP5K1 inhibitor is a PIP5K1 selective inhibitor.
The engineered cell of any one of claims 56-57, wherein said PIP5K1 inhibitor is ISA2011B, a dominant-negative PIP5K1, and/or derivatives thereof.
The engineered cell of claim 58, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199 mutant and/or a PIP5K1 L207 mutant.
The engineered cell of any one of claims 58-59, wherein said dominant-negative PIP5K1 comprises a PIP5K1 L199I mutant and/or a PIP5K1 L207I mutant.
A method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of Rab35 in said cell.
The method of claim 61, wherein said regulating the expression and/or function of Rab35 comprises providing and/or overexpressing said Rab35 in said cell.
The method of claim 62, wherein said regulating the expression and/or function of Rab35 comprises providing and/or overexpressing a constitutively active form of Rab35 in said cell.
The method of claim 63, wherein said constitutively active form of Rab35 is Rab35 Q67 mutant.
The method of any one of claims 63-64, wherein said constitutively active form of Rab35 is Rab35 Q67L mutant.
The method of claim 62, wherein said regulating the expression and/or function of Rab35 comprises knocking out or knocking down the expression of a gene encoding for said Rab35 in said cell.
The method of claim 62, wherein said regulating the expression and/or function of Rab35 comprises administering a Rab35 inhibitor to said cell.
The method of claim 67, wherein said Rab35 inhibitor is a Rab35 selective inhibitor.
The method of any one of claims 67-68, wherein said Rab35 inhibitor is a Rab35 antibody, a dominant-negative Rab35, and/or derivatives thereof.
The method of claim 69, wherein said dominant-negative Rab35 comprises a Rab35 S22 mutant.
The method of any one of claims 69-70, wherein said dominant-negative Rab35 comprises a Rab35 S22N mutant.
The method of any one of claims 69-71, wherein said dominant-negative Rab35 comprises a Rab35 mutant without positively charged residue.
The method of any one of claims 69-72, wherein said dominant-negative Rab35 comprises a Rab35 mutant with negatively charged Glutamic acid residue.
An engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of Rab35 therein.
The engineered cell of claim 74, said engineered cell has been modified to provide and/or overexpress said Rab35.
The engineered cell of any one of claims 74-75, said engineered cell has been modified to provide and/or overexpress Rab35 Q67 mutant.
The engineered cell of any one of claims 74-76, said engineered cell has been modified to provide and/or overexpress Rab35 Q67L mutant.
The engineered cell of claim 74, wherein the expression of a gene encoding for said Rab35 has been knocked out or knocked down.
The engineered cell of claim 74, said engineered cell has been treated with a Rab35 inhibitor.
The engineered cell of claim 79, wherein said Rab35 inhibitor is a Rab35 selective inhibitor.
The engineered cell of any one of claims 79-80, wherein said Rab35 inhibitor is a Rab35 antibody, a dominant-negative Rab35, and/or derivatives thereof.
The engineered cell of claim 81, wherein said dominant-negative Rab35 comprises a Rab35 S22 mutant.
The engineered cell of any one of claims 81-82, wherein said dominant-negative Rab35 comprises a Rab35 S22N mutant.
The engineered cell of any one of claims 81-83, wherein said dominant-negative Rab35 comprises a Rab35 mutant without positively charged residue.
The engineered cell of any one of claims 81-84, wherein said dominant-negative Rab35 comprises a Rab35 mutant with negatively charged Glutamic acid residue.
A method for regulating migrasome formation by a cell and/or migrasome-mediated biological process, comprising regulating the amount and/or function of integrin α5 (ITGa5) in said cell.
The method of claim 86, wherein said regulating the expression and/or function of ITGa5 comprises providing and/or overexpressing said ITGa5 in said cell.
The method of any one of claims 86-87, wherein said regulating the expression and/or function of ITGa5 comprises providing and/or overexpressing a cytosolic domain of ITGa5 in said cell.
The method of any one of claims 86-88, wherein said regulating the expression and/or function of ITGa5 comprises providing and/or overexpressing GFFKR motif of cytosolic domain of ITGa5 in said cell.
The method of claim 86, wherein said method comprises providing and/or overexpressing ITGa5 and Rab35 binding protein in said cell.
The method of claim 86, wherein said regulating the expression and/or function of ITGa5 comprises knocking out or knocking down the expression of a gene encoding for said ITGa5 in said cell.
The method of claim 86, wherein said method comprises providing and/or overexpressing ITGa5 with no Rab35 binding in said cell.
The method of claim 86, wherein said regulating the expression and/or function of ITGa5 comprises administering a ITGa5 inhibitor to said cell.
The method of claim 93, wherein said ITGa5 inhibitor is a ITGa5 selective inhibitor.
The method of any one of claims 93-94, wherein said ITGa5 inhibitor is a ITGa5 antibody, a dominant-negative ITGa5, and/or derivatives thereof.
The method of claim 95, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant without cytosolic domain.
The method of any one of claims 95-96, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant without GFFKR motif.
The method of any one of claims 95-97, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant in which GFFKR is mutated.
The method of any one of claims 95-98, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant in which GFFKR is mutated to AAAAA.
An engineered cell with altered ability for forming migrasomes comparing to a corresponding unmodified cell, said engineered cell has been modified to alter the amount and/or function of ITGa5 therein.
The engineered cell of claim 100, said engineered cell has been modified to provide and/or overexpress said ITGa5.
The engineered cell of any one of claims 100-101, said engineered cell has been modified to provide and/or overexpress a cytosolic domain of ITGa5.
The engineered cell of any one of claims 100-102, said engineered cell has been modified to provide and/or overexpress GFFKR motif of cytosolic domain of ITGa5.
The engineered cell of claim 100, said engineered cell has been modified to provide and/or overexpress ITGa5 and Rab35 binding protein.
The engineered cell of claim 100, wherein the expression of a gene encoding for said ITGa5 has been knocked out or knocked down.
The engineered cell of claim 100, wherein said engineered cell has been modified to provide and/or overexpress ITGa5 with no Rab35 binding.
The engineered cell of claim 100, said engineered cell has been treated with a ITGa5 inhibitor.
The engineered cell of claim 107, wherein said ITGa5 inhibitor is a ITGa5 selective inhibitor.
The engineered cell of any one of claims 107-108, wherein said ITGa5 inhibitor is a ITGa5 antibody, a dominant-negative ITGa5, and/or derivatives thereof.
The engineered cell of claim 109, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant without cytosolic domain.
The engineered cell of any one of claims 109-110, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant without GFFKR motif.
The engineered cell of any one of claims 109-111, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant in which GFFKR is mutated.
The engineered cell of any one of claims 109-112, wherein said dominant-negative ITGa5 comprises a ITGa5 mutant in which GFFKR is mutated to AAAAA.