Dataset Information

CYRI-B loss promotes enlarged mature focal adhesions and restricts microtubule and ERC1 access to the cell leading edge

ABSTRACT: CYRI proteins promote lamellipodial dynamics by opposing Rac1-mediated activation of the Scar/WAVE complex. This activity also supports resolution of macropinocytic cups, promoting internalisation of surface proteins, including integrins. Here, we show that CYRI-B also promotes focal adhesion maturation and dynamics. Focal adhesions in CYRI-B-depleted cells show accelerated maturation and become excessively large. We probed the composition of these enlarged focal adhesions, using a Bio-ID screen, with paxillin as bait. Our screen revealed changes in adhesion proteins proximal to paxillin suggesting early activation of stress fibre contraction and depletion of the integrin internalisation mediator ERC1. Lack of CYRI-B leads to more stable lamellipodia and accumulation of polymerised actin in stress fibres. This actin acts as a barrier to microtubule targeting for adhesion turnover. Thus, our studies reveal an important connection between lamellipodia dynamics controlled by CYRI-B and microtubule targeting of ERC1 to modulate adhesion maturation and turnover.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Melanocyte

DISEASE(S): Skin Melanoma

SUBMITTER: Sergio Lilla

LAB HEAD: Laura Machesky

PROVIDER: PXD062975 | Pride | 2025-12-29

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20190530_SEL_LU_JAW_R02_290519_Fam49A-KO_plus-Bio_016.raw	Raw
	20190530_SEL_LU_JAW_R02_290519_Fam49A-KO_plus-Bio_017.raw	Raw
	20190530_SEL_LU_JAW_R02_290519_Fam49A-KO_plus-Bio_018.raw	Raw
	20190530_SEL_LU_JAW_R02_290519_Fam49B-KO_minus-Bio_013.raw	Raw
	20190530_SEL_LU_JAW_R02_290519_Fam49B-KO_minus-Bio_014.raw	Raw

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Publications

CYRI-B loss promotes enlarged mature focal adhesions and restricts microtubule and ERC1 access to the cell leading edge.

Whitelaw Jamie A JA Ghosh Sayantika S Lilla Sergio S Nikolaou Savvas S Tweedy Luke L Fort Loic L Paul Nikki R NR Zanivan Sara S Gadegaard Nikolaj N Insall Robert H RH Machesky Laura M LM

Journal of cell science 20251124 22

CYRI proteins promote lamellipodial dynamics by opposing Rac1-mediated activation of the Scar/WAVE complex. This activity also supports resolution of macropinocytic cups, promoting internalisation of surface proteins, including integrins. Here, we show that CYRI-B also promotes focal adhesion maturation and dynamics. Focal adhesions in CYRI-B-depleted cells show accelerated maturation and become excessively large. We probed the composition of these enlarged focal adhesions, using a Bio-ID screen ...[more]

PMID: 41277545

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Project description:Introduction Metastatic potential and rising incidence make cutaneous melanoma a major health concern worldwide. Although the therapies currently in use have proven effective, their use is limited by side effects arising and development of drug resistance. For this reason, research for new therapeutic targets and predictive biomarkers is crucial. Semaphorin 5A (SEMA5A) is a member of the Semaphorin family with involvement in cancer biology and neural development. We previously demonstrated that this protein promotes different in vitro melanoma aggressive features. In this work we further investigated the role and the mechanism of action of SEMA5A in melanoma formation and progression. Methods We performed proteomic analysis of stable knockdown SEMA5A human melanoma cells, and in silico analysis to identify cellular pathways modulated by SEMA5A. In vitro clonogenic assay and cell viability assay under different substrate conditions were conducted by using both human and murine melanoma SEMA5A knockdown clones. Western blotting analysis and immunofluorescence have been employed to investigate focal adhesion pathway regulation and lamellipodia formation after SEMA5A depletion. Nocodazole assay was used to evaluate focal adhesion dynamic. Xenograft mouse model of melanoma was used to investigate the effect of SEMA5A depletion in tumor formation and growth. Results Ingenuity pathway analysis showed that SEMA5A effects several different cellular pathways essential for cell viability, cell proliferation, and cytoskeletal dynamics, including focal adhesion and lamellipodia formation. We demonstrated that SEMA5A depletion reduced clonogenic properties and cell viability of cells in both low and high stiffness condition of culture. SEMA5A affected Focal adhesion kinase and Paxillin phosphorylation, integrin beta 1 activation, thus regulating focal adhesion dynamics, and lamellipodia formation, regulating Arp3 nucleation and expression. Delay of tumor formation and reduced tumor growth was observed in mice bearing tumor expressing low level of SEMA5A. Conclusions These findings demonstrate that SEMA5A is a novel player of melanoma aggressiveness and progression, being involved in modulating cell viability, focal adhesion signaling and lamellipodia in vitro and tumor growth in vivo.