light-induced-molecular-adsorption-proteins-using-primo-system-for

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

The University of Manchester

&#x7C98;&#x7740; (FA) &#x62C6;&#x5378;&#x6240;&#x9700;&#x7684;&#x6700;&#x4F73;&#x7EC6;&#x80DE;&#x8FC1;&#x79FB;&#x88AB;&#x4ECB;&#x5BFC;&#x5FAE;&#x7BA1; (MTs) &#xFF1B;&#x9488;&#x5BF9;&#x7531; MTs FAs &#x6B63;&#x503C;&#x5176;&#x53CD;&#x6C47;&#x7F16;&#x3002;MTs &#x518D;&#x751F;&#x8BF1;&#x5BFC;&#x7684; MT destabilizer nocodazole&#xFF0C;&#x53BB;&#x9664;&#x6FC0;&#x6D3B; Rho &#x6837;&#x86CB;&#x767D; Rac&#xFF0C;&#x4F34;&#x968F; FA &#x53CD;&#x6C47;&#x7F16;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x8868;&#x660E;&#xFF0C;Rac &#x9E1F;&#x560C;&#x5464;&#x6838;&#x82F7;&#x9178;&#x4EA4;&#x6362;&#x56E0;&#x5B50; &#xFF08;&#x5168;&#x7403;&#x57FA;&#x91D1;&#xFF09; Sif &#x548C; Tiam1 &#x7C7B;&#x4F3C;&#x4EA4;&#x6D41;&#x56E0;&#x7D20; &#xFF08;&#x5230;&#x59FF;&#x59FF;) &#x662F;&#x8D1F;&#x8D23; Rac &#x6FC0;&#x6D3B;&#x671F;&#x95F4;&#x5C71;&#x518D;&#x751F;&#x3002;&#x91CD;&#x8981;&#x7684;&#x662F;&#xFF0C;&#x9700;&#x8981;&#x591A;&#x4E2A;&#x76EE;&#x6807;&#x7684; FAs &#x7684; MTs &#x5230;&#x59FF;&#x59FF;&#x3002;&#x5176;&#x7ED3;&#x679C;&#x662F;&#xFF0C;&#x5230;&#x59FF;&#x59FF;&#x7EC4;&#x5408;&#x5F0F;&#x7EC6;&#x80DE; FAs &#x6709;&#x51CF;&#x5C11;&#x7684;&#x62C6;&#x5378;&#x7387;&#xFF0C;&#x56E0;&#x6B64;&#x653E;&#x5927;&#x3002;&#x8FD9;&#x5C31;&#x5BFC;&#x81F4;&#x8FC1;&#x79FB;&#x7684;&#x51CF;&#x5C11;&#x901F;&#x5EA6;&#x3002;&#x5728;&#x4E00;&#x8D77;&#xFF0C;&#x8FD9;&#x4E9B;&#x8C03;&#x67E5;&#x7ED3;&#x679C;&#x5EFA;&#x8BAE;&#x4E00;&#x4E2A;&#x65B0;&#x7684;&#x89D2;&#x8272;&#x5230;&#x59FF;&#x59FF; FA &#x53CD;&#x6C47;&#x7F16;&#x548C;&#x7EC6;&#x80DE;&#x8FC1;&#x79FB;&#x901A;&#x8FC7;&#x4ECB;&#x5BFC;&#x5C71;&#x673A;&#x5236;&#x3002;

Rac &#x6FC0;&#x6D3B;&#x5242;&#x5230;&#x59FF;&#x59FF; &#xFF08;Tiam2&#xFF09; &#x7531;&#x5FAE;&#x7BA1;&#x4ECB;&#x5BFC;&#x7C98;&#x7740;&#x62C6;&#x5378;&#x8C03;&#x8282;&#x7EC6;&#x80DE;&#x8FC1;&#x79FB;&#x3002;

Focal adhesion (FA) disassembly required for optimal cell migration is mediated by microtubules (MTs); targeting of FAs by MTs coincides with their disassembly. Regrowth of MTs, induced by removal of the MT destabilizer nocodazole, activates the Rho-like GTPase Rac, concomitant with FA disassembly. Here, we show that the Rac guanine nucleotide exchange factor (GEF) Sif and Tiam1-like exchange factor (STEF) is responsible for Rac activation during MT regrowth. Importantly, STEF is required for multiple targeting of FAs by MTs. As a result, FAs in STEF-knockdown cells have a reduced disassembly rate and are consequently enlarged. This leads to reduced speed of migration. Together, these findings suggest a new role for STEF in FA disassembly and cell migration through MT-mediated mechanisms.

The Rac activator STEF (Tiam2) regulates cell migration by microtubule-mediated focal adhesion disassembly.

D&#x26;#39;adh&#xE9;sion focale (FA) d&#xE9;montage n&#x26;#39;est n&#xE9;cessaire pour la migration cellulaire optimale est m&#xE9;di&#xE9;e par les microtubules (MT), le ciblage des AF par MTS co&#xEF;ncide avec leur d&#xE9;montage. Repousse des MT, induite par &#xE9;limination du nocodazole MT d&#xE9;stabilisant, active le GTPase de type Rho Rac, concomitante avec FA d&#xE9;montage. Ici, nous montrons que le Rac nucl&#xE9;otide guanine facteur d&#x26;#39;&#xE9;change (FEM) et Sif-Tiam1 comme facteur d&#x26;#39;&#xE9;change (STEF) est responsable de l&#x26;#39;activation Rac au cours MT repousse. Il est important, STEF est n&#xE9;cessaire pour le ciblage multiple de AF par MTS. En cons&#xE9;quence, les autorit&#xE9;s f&#xE9;d&#xE9;rales dans STEF-knockdown cellules ont un taux r&#xE9;duit de d&#xE9;montage et sont par cons&#xE9;quent &#xE9;largie. Cela conduit &#xE0; une vitesse r&#xE9;duite de la migration. Ensemble, ces r&#xE9;sultats sugg&#xE8;rent un nouveau r&#xF4;le pour STEF en FA d&#xE9;montage et la migration cellulaire par le biais des m&#xE9;canismes de m&#xE9;diation MT-.

Le Rac Activator STEF (Tiam2) R&#xE9;glemente la migration cellulaire par des microtubules induite par Focal Adhesion D&#xE9;montage

Fokale Adh&auml;sion (FA) Demontage erforderlich f&uuml;r optimale Zellmigration ist vermittelt durch Mikrotubuli (MTs); Ausrichtung der FAs von MTs f&auml;llt mit ihrer Demontage. Nachwachsen von MTs, induziert durch Abbau der MT Destabilizer Nocodazole, aktiviert die Rho-&auml;hnlichen GTPase Rac, gleichzeitige mit FA-Demontage. Hier zeigen wir, dass den Rac-Guanin-Nukleotid-Austausch (GEF) Sif und Tiam1-wie Exchange-Faktors Faktor (STEF) ist verantwortlich f&uuml;r Rac-Aktivierung w&auml;hrend MT nachwachsen. STEF ist vor allem f&uuml;r mehrere Angriffe auf FAs von MTs erforderlich. Infolgedessen FAs in STEF-Niederschlag-Zellen haben eine reduzierte Demontage und werden folglich vergr&ouml;&szlig;ert. Dies f&uuml;hrt zu verminderter Geschwindigkeit der Migration. Zusammen, lassen diese Ergebnisse eine neue Rolle f&uuml;r STEF FA Demontage und Zellmigration durch MT-vermittelten Mechanismen.

Der Rac-Aktivator STEF (Tiam2) reguliert Zellmigration durch fokale Adh&auml;sion Microtubule-vermittelte Demontage.

Smontaggio di adesione focale (FA) necessario per la migrazione cellulare ottimale &egrave; mediato da microtubuli (MTs); targeting di FAs da MTs coincide con il loro smontaggio. Ricrescita di MTs, indotta dalla rimozione del nocodazole destabilizer MT, attiva il GTPase Rac Rho-like, concomitante con smontaggio FA. Qui, ci dimostrano che lo scambio di nucleotide della guanina Rac fattore (GEF) Sif e scambio Tiam1-come fattore (STEF) &egrave; responsabile per l&#39;attivazione di Rac durante la ricrescita di MT. Soprattutto, STEF &egrave; richiesto per il targeting pi&ugrave; di FAs da MTs. Di conseguenza, FAs nelle cellule STEF-atterramento hanno un tasso ridotto di smontaggio e di conseguenza sono ingrossati. Questo porta a ridotta velocit&agrave; di migrazione. Insieme, questi risultati suggeriscono un ruolo nuovo per STEF in FA smontaggio e migrazione cellulare attraverso meccanismi mediati da MT.

L&#39;attivatore Rac STEF (Tiam2) regola la migrazione cellulare da smontaggio di adesione focale microtubule-mediata.

&#x5206;&#x89E3;&#x306B;&#x6700;&#x9069;&#x306A;&#x7D30;&#x80DE;&#x79FB;&#x52D5;&#x306B;&#x5FC5;&#x8981;&#x306A;&#x63A5;&#x7740;&#x6591;&#xFF08;FA&#xFF09;&#x306F;&#x3001;&#x5FAE;&#x5C0F;&#x7BA1;&#xFF08;MTS&#xFF09;&#x306B;&#x3088;&#x3063;&#x3066;&#x5A92;&#x4ECB;&#x3055;&#x308C;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;MTS&#x306B;&#x3088;&#x3063;&#x3066;Fas&#x306E;&#x6A19;&#x7684;&#x306F;&#x3001;&#x305D;&#x308C;&#x3089;&#x306E;&#x5206;&#x89E3;&#x3068;&#x4E00;&#x81F4;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002; MT destabilizer&#x30CE;&#x30B3;&#x30C0;&#x30BE;&#x30FC;&#x30EB;&#x306E;&#x9664;&#x53BB;&#x306B;&#x3088;&#x3063;&#x3066;&#x8A98;&#x5C0E;&#x3055;&#x308C;&#x308B;MT&#x306E;&#x518D;&#x751F;&#x306B;&#x306F;&#x3001;GTP&#x30A2;&#x30FC;&#x30BC;&#x306E;Rac&#x3001;FA&#x5206;&#x89E3;&#x3068;&#x306E;&#x4F75;&#x7528;&#x306E;Rho&#x306E;&#x3088;&#x3046;&#x306A;&#x30A2;&#x30AF;&#x30C6;&#x30A3;&#x30D6;&#x306B;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;Rac&#x306E;&#x30B0;&#x30A2;&#x30CB;&#x30F3;&#x30CC;&#x30AF;&#x30EC;&#x30AA;&#x30C1;&#x30C9;&#x4EA4;&#x63DB;&#x56E0;&#x5B50;&#xFF08;GEF&#xFF09;SIF&#x3068;&#x4EA4;&#x63DB;&#x56E0;&#x5B50;Tiam1&#x306E;&#x3088;&#x3046;&#x306A;&#xFF08;STEF&#xFF09;&#x306F;MT&#x518D;&#x751F;&#x6642;&#x306E;Rac&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x8CAC;&#x4EFB;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x91CD;&#x8981;&#x306A;&#x306E;&#x306F;&#x3001;STEF&#x306F;&#x3001;MTS&#x306B;&#x3088;&#x3063;&#x3066;FA&#x306E;&#x8907;&#x6570;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x304C;&#x5FC5;&#x8981;&#x3068;&#x306A;&#x308A;&#x307E;&#x3059;&#x3002;&#x305D;&#x306E;&#x7D50;&#x679C;&#x3001;STEF-&#x30CE;&#x30C3;&#x30AF;&#x30C0;&#x30A6;&#x30F3;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;Fas&#x306F;&#x3001;&#x9084;&#x5143;&#x5206;&#x89E3;&#x7387;&#x3092;&#x6301;&#x3063;&#x3066;&#x3001;&#x305D;&#x306E;&#x7D50;&#x679C;&#x3001;&#x62E1;&#x5927;&#x3055;&#x308C;&#x307E;&#x3059;&#x3002;&#x3053;&#x308C;&#x306F;&#x3001;&#x79FB;&#x884C;&#x306E;&#x6E1B;&#x901F;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B;&#x3002;&#x4E00;&#x7DD2;&#x306B;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x77E5;&#x898B;&#x306F;&#x3001;MT-&#x5A92;&#x4ECB;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x4ECB;&#x3057;&#x3066;FA&#x5206;&#x89E3;&#x3068;&#x7D30;&#x80DE;&#x904A;&#x8D70;&#x306E;STEF&#x306E;&#x65B0;&#x305F;&#x306A;&#x5F79;&#x5272;&#x3092;&#x793A;&#x5506;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

Rac&#x306E;&#x6D3B;&#x6027;&#x5316;&#x56E0;&#x5B50;STEF&#xFF08;Tiam2&#xFF09;&#x306F;&#x3001;&#x5FAE;&#x5C0F;&#x7BA1;&#x5206;&#x89E3;&#x4ECB;&#x5728;&#x63A5;&#x7740;&#x6591;&#x306B;&#x3088;&#x308B;&#x7D30;&#x80DE;&#x904A;&#x8D70;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;

&#xCD5C;&#xC801;&#xC758; &#xC140; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158;&#xC5D0; &#xD544;&#xC694;&#xD55C; &#xCD08;&#xC810; &#xC811;&#xCC29; (FA) &#xD574;&#xCCB4; microtubules (MTs); &#xC911;&#xC7AC; Mts&#xC5D0; &#xC758;&#xD574; Fas&#xC758; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xADF8;&#xB4E4;&#xC758; &#xD574;&#xCCB4;&#xC640; &#xC77C;&#xCE58; &#xD55C;&#xB2E4;. Mts&#xC758; Regrowth, MT destabilizer nocodazole&#xC758; &#xC81C;&#xAC70;&#xC5D0; &#xC758;&#xD574; &#xC720;&#xB3C4; &#xB41C; FA &#xD574;&#xCCB4;&#xC640; &#xBD80;&#xC218;&#xC801;&#xC778; Rho &#xAC19;&#xC740; GTPase Rac &#xD65C;&#xC131;&#xD654; &#xB429;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xAC00; &#xBCF4;&#xC5EC;&#xC8FC;&#xB294; Rac guanine &#xC5FC;&#xAE30; &#xAD50;&#xD658; (GEF) Sif &#xBC0F; Tiam1 &#xAC19;&#xC740; exchange &#xC694;&#xC18C; &#xC694;&#xC18C; (STEF)&#xB294; &#xC790;&#xB77C;&#xB098;&#xB294; &#xC0B0; &#xC911;&#xC5D0; Rac &#xD65C;&#xC131;&#xD654;&#xC5D0; &#xB300; &#xD55C; &#xCC45;&#xC784;. &#xC911;&#xC694; &#xD55C; &#xAC83;&#xC740;, STEF&#xB294; Mts&#xC5D0; &#xC758;&#xD574; Fas&#xC758; &#xB2E4;&#xC911; &#xB300;&#xC0C1; &#xC9C0;&#xC815;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD569;&#xB2C8;&#xB2E4;. &#xADF8; &#xACB0;&#xACFC;, FAs STEF &#xCD5C;&#xC800;&#xC758; &#xC140;&#xC5D0;&#xC11C; &#xAC10;&#xC18C; &#xBD84;&#xD574; &#xC18D;&#xB3C4; &#xC788;&#xACE0; &#xACB0;&#xACFC;&#xC801;&#xC73C;&#xB85C; &#xD655;&#xB300;. &#xC774; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158;&#xC758; &#xAC10;&#xC18C; &#xC18D;&#xB3C4;&#xB97C; &#xB048;&#xB2E4;. &#xD568;&#xAED8;, &#xC774;&#xB7EC;&#xD55C; &#xC5F0;&#xAD6C; &#xACB0;&#xACFC; &#xC81C;&#xC548; &#xC0C8;&#xB85C;&#xC6B4; &#xC5ED;&#xD560; STEF FA &#xD574;&#xCCB4; &#xBC0F; &#xC140; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158; MT &#xC911;&#xC7AC; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xD1B5;&#xD574;.

Rac &#xD65C;&#xC131;&#xAE30; STEF (Tiam2) &#xCD08;&#xC810; &#xC811;&#xCC29; microtubule &#xC911;&#xC7AC; &#xBD84;&#xD574; &#xD558; &#xC5EC; &#xC140; &#xB9C8;&#xC774;&#xADF8;&#xB808;&#xC774;&#xC158;&#xC744; &#xADDC;&#xC81C; &#xD55C;&#xB2E4;.

Adhesi&#xF3;n focal (FA), el desmontaje se requiere para la migraci&#xF3;n de c&#xE9;lulas &#xF3;ptimo es mediado por microt&#xFA;bulos (MT), focalizaci&#xF3;n de los combustibles por las MTs coincide con su desmontaje. El rebrote de las MTs, inducida por la privaci&#xF3;n de la nocodazole MT desestabilizador, se activa la Rho-GTPasa Rac, como, concomitante con la FA desmontaje. Aqu&#xED;, mostramos que el intercambio de nucle&#xF3;tidos de guanina Rac factor (FMAM) y Sif Tiam1-como factor de cambio (STEF) es responsable de la activaci&#xF3;n de Rac en MT rebrote. Es importante destacar que, STEF es necesaria para la focalizaci&#xF3;n m&#xFA;ltiple de AF por MTS. Como resultado, AF en las c&#xE9;lulas de STEF-desmontables tienen una tasa reducida y el desmontaje se agrandan en consecuencia. Esto conduce a una velocidad reducida de la migraci&#xF3;n. En conjunto, estos hallazgos sugieren un nuevo papel para el desmontaje STEF en la FA y la migraci&#xF3;n celular a trav&#xE9;s de mecanismos mediados por MT.

El RAC Activador STEF (Tiam2) Regula la migraci&#xF3;n de las c&#xE9;lulas por los microt&#xFA;bulos mediada por adhesi&#xF3;n focal desmontaje

Focal adhesions (FAs) control cell shape and motility, which are important processes that underlie a wide range of physiological functions. FA dynamics is regulated by cytoskeleton, motor proteins and small GTPases. Kinectin is an integral endoplasmic reticulum (ER) membrane protein that extends the ER along microtubules. Here, we investigated the influence of the ER on FA dynamics within the cellular lamella by disrupting the kinectin-kinesin interaction by overexpressing the minimal kinectin-kinesin interaction domain on kinectin in cells. This perturbation resulted in a morphological change to a rounded cell shape and reduced cell spreading and migration. Immunofluorescence and live-cell imaging demonstrated a kinectin-dependent ER extension into the cellular lamella and ER colocalisation with FAs within the cellular lamella. FRAP experiments showed that ER contact with FAs was accompanied with an increase in FA protein recruitment to FAs. Disruption of the kinectin-kinesin interaction caused a reduction in FA protein recruitment to FAs. This suggests that the ER supports FA growth within the cellular lamella. Microtubule targeting to FAs is known to promote adhesion disassembly; however, ER contact increased FA size even in the presence of microtubules. Our results suggest a scenario whereby kinectin-kinesin interaction facilitates ER transport along microtubules to support FA growth.

Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella.

This chapter describes the use of microscope-based fluorescence recovery after photobleaching (FRAP). To quantify the dynamics of proteins within a subcellular compartment, we first outline the general aspects of FRAP experiments and then provide a detailed protocol of how to measure and analyse the most important parameters of FRAP experiments such as mobile fraction and half-time of recovery.

Fluorescence recovery after photobleaching.

Scanning acoustic microscopy is potentially a powerful tool for characterizing the elastic properties of soft biological tissues and cells. In this paper, we present a method, multi-layer phase analysis (MLPA), which can be used to extract local speed of sound values, for both thin tissue sections mounted on glass slides and cultured cells grown on cell culture plastic, with a resolution close to 1 μm. The method exploits the phase information that is preserved in the interference between the acoustic wave reflected from the substrate surface and internal reflections from the acoustic lens. In practice, a stack of acoustic images are captured beginning with the acoustic focal point 4 μm above the substrate surface and moving down in 0.1-μm increments. Scanning parameters, such as acoustic wave frequency and gate position, were adjusted to obtain optimal phase and lateral resolution. The data were processed offline to extract the phase information with the contribution of any inclination in the substrate removed before the calculation of sound speed. Here, we apply this approach to both skin sections and fibroblast cells, and compare our data with the V(f) (voltage versus frequency) method that has previously been used for characterization of soft tissues and cells. Compared with the V(f) method, the MPLA method not only reduces signal noise but can be implemented without making a priori assumptions with regards to tissue or cell parameters.

Multi-layer phase analysis: quantifying the elastic properties of soft tissues and live cells with ultra-high-frequency scanning acoustic microscopy.

The correct outgrowth of axons is essential for the development and regeneration of nervous systems. Axon growth is primarily driven by microtubules. Key regulators of microtubules in this context are the spectraplakins, a family of evolutionarily conserved actin-microtubule linkers. Loss of function of the mouse spectraplakin ACF7 or of its close Drosophila homolog Short stop/Shot similarly cause severe axon shortening and microtubule disorganization. How spectraplakins perform these functions is not known. Here we show that axonal growth-promoting roles of Shot require interaction with EB1 (End binding protein) at polymerizing plus ends of microtubules. We show that binding of Shot to EB1 requires SxIP motifs in Shot's C-terminal tail (Ctail), mutations of these motifs abolish Shot functions in axonal growth, loss of EB1 function phenocopies Shot loss, and genetic interaction studies reveal strong functional links between Shot and EB1 in axonal growth and microtubule organization. In addition, we report that Shot localizes along microtubule shafts and stabilizes them against pharmacologically induced depolymerization. This function is EB1-independent but requires net positive charges within Ctail which essentially contribute to the microtubule shaft association of Shot. Therefore, spectraplakins are true members of two important classes of neuronal microtubule regulating proteins: +TIPs (tip interacting proteins; plus end regulators) and structural MAPs (microtubule-associated proteins). From our data we deduce a model that relates the different features of the spectraplakin C terminus to the two functions of Shot during axonal growth.

Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins).

Crosstalk between the microtubule (MT) and actin cytoskeletons is fundamental to many cellular processes including cell polarisation and cell motility. Previous work has shown that members of the growth-arrest-specific 2 (GAS2) family mediate the crosstalk between filamentous actin (F-actin) and MTs, but the molecular basis of this process remained unclear. By using fluorescence microscopy, we demonstrate that three members of this family, GAS2-like 1, GAS2-like 2 and GAS2-like 3 (G2L1, G2L2 and G2L3, also known as GAS2L1, GAS2L2 and GAS2L3, respectively) are differentially involved in mediating the crosstalk between F-actin and MTs. Although all localise to actin and MTs, only the exogenous expression of G2L1 and G2L2 influenced MT stability, dynamics and guidance along actin stress fibres. Biochemical analysis and live-cell imaging revealed that their functions are largely due to the association of these proteins with MT plus-end-binding proteins that bind to SxIP or SxLP motifs located at G2L C-termini. Our findings lead to a model in which end-binding (EB) proteins play a key role in mediating actin-MT crosstalk. 

GAS2-like proteins mediate communication between microtubules and actin through interactions with end-binding proteins.

External forces play a key role in shaping development and normal physiology. Aberrant responses to forces, or changes in the nature of such forces, are implicated in a variety of diseases. Cells contain several types of adhesions, linking them to their external environment. It is through these adhesions that forces are both sensed (from the outside inwards) and applied (from inside to out). Furthermore, several adhesion-based proteins are sensitive to changes in intracellular forces, utilising them for activation and regulation. Here, we outline how vinculin, a key component of integrin-mediated adhesions linking the actin cytoskeleton to the extracellular matrix (ECM), is regulated by force and acts as force transducing protein. We discuss the role of vinculin in vivo and its place in health and disease; summarise the proposed mechanisms by which vinculin is recruited to and activated at integrin-ECM adhesions; and discuss recent findings that place vinculin as the major force sensing and transmitting component of cell-matrix adhesion complexes. Finally, we discuss the role of vinculin in regulating the cellular responses to both the physical properties of the external environment and to externally applied physical stimuli.

Mechanosensitive components of integrin adhesions: Role of vinculin.

Focal adhesions (FAs) are macromolecular complexes that regulate cell adhesion and mechanotransduction. By performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoactivation (FLAP) experiments, we found that the mobility of core FA proteins correlates with their function. Structural proteins such as tensin, talin and vinculin are significantly less mobile in FAs than signaling proteins such as FAK (also known as PTK2) and paxillin. The mobilities of the structural proteins are directly influenced by substrate stiffness, suggesting that they are involved in sensing the rigidity of the extracellular environment. The turnover rates of FAK and paxillin, as well as kindlin2 (also known as FERMT2), are not influenced by substrate stiffness. By using specific Src and FAK inhibitors, we reveal that force-sensing by vinculin occurs independently of FAK and paxillin phosphorylation. However, their phosphorylation is required for downstream Rac1-driven cellular processes, such as protrusion and cell migration. Overall, we show that the FA is composed of different functional modules that separately control mechanosensing and the cellular mechano-response.

Distinct focal adhesion protein modules control different aspects of mechanotransduction.

Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUS-induced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity.

The ability of cells to sense and respond to mechanical signals is vital in development and healthy tissue functioning. Many diseases are related to either changing mechanical properties of the tissue, or changes in the ability of cells to sense mechanical signals. This sensing occurs, in part, at integrin-associated complexes (IACs) that form sites of attachment between the cell and the extracellular matrix (ECM). In this review, we discuss the complex mechanical signals of the ECM. We will also outline how IACs are involved in cellular sensing of these mechanical properties, focussing on the molecular mechanisms of key adhesion molecules. Finally, the cellular mechanisms of mechanotransduction considering mechanosensing and signalling aspects of the core proteins in FAs are discussed and open questions outlined.

Mechanotransduction at the cell-matrix interface.

Cell-matrix interactions and podocyte intercellular junctions are key for maintaining the glomerular filtration barrier. Vinculin, a cytoplasmic protein, couples actin filaments to integrin-mediated cell-matrix adhesions and to cadherin-based intercellular junctions. Here, we examined the role of vinculin in podocytes by the generation of a podocyte-specific knockout mouse. Mice lacking podocyte vinculin had increased albuminuria and foot process effacement following injury in vivo. Analysis of primary podocytes isolated from the mutant mice revealed defects in cell protrusions, altered focal adhesion size and signaling, as well as impaired cell migration. Furthermore, we found a marked mislocalization of the intercellular junction protein zonula occludens-1. In kidney sections from patients with focal segmental glomerulosclerosis, minimal change disease and membranous nephropathy, we observed dramatic differences in the expression levels and localization of vinculin. Thus, our results suggest that vinculin is necessary to maintain the integrity of the glomerular filtration barrier by modulating podocyte foot processes and stabilizing intercellular junctions.

Vinculin is required to maintain glomerular barrier integrity.

Cell division involves the tightly coordinated rearrangement of actin and microtubules (MTs). We have previously shown that a member of the family of growth arrest-specific 2-like proteins, GAS2-like 1 (G2L1) regulates actin-MT crosstalk through its associations with plus-end microtubule tip-binding (EB) proteins. Here we show that G2L1 is involved in the regulation of cell division. We show that the depletion of G2L1 results in a reduction in the number of cells undergoing cell division and a significant proportion of those cells that do divide are either multinucleated, display deformed nuclei, or undergo cell division at a much slower rate. Exogenous expression of G2L1 mutants revealed that the association of G2L1 with EB1 is critical for regulated cell division and blocking this interaction inhibits cell division as observed in cells lacking G2L1. Taken together, our data suggest that G2L1 controls the precise regulation and successful progression of cell division through its binding to EB-proteins.

Article	Year
The Rac activator STEF (Tiam2) regulates cell migration by microtubule-mediated focal adhesion disassembly. EMBO reports\| PubMed ID: 20224579	2010
Kinectin-mediated endoplasmic reticulum dynamics supports focal adhesion growth in the cellular lamella. Journal of cell science\| PubMed ID: 20980389	2010
Fluorescence recovery after photobleaching. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 21748690	2011
Multi-layer phase analysis: quantifying the elastic properties of soft tissues and live cells with ultra-high-frequency scanning acoustic microscopy. IEEE transactions on ultrasonics, ferroelectrics, and frequency control\| PubMed ID: 22547273	2012
Spectraplakins promote microtubule-mediated axonal growth by functioning as structural microtubule-associated proteins and EB1-dependent +TIPs (tip interacting proteins). The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 22764224	2012
GAS2-like proteins mediate communication between microtubules and actin through interactions with end-binding proteins. Journal of cell science\| PubMed ID: 24706950	2014
Mechanosensitive components of integrin adhesions: Role of vinculin. Experimental cell research\| PubMed ID: 26607713	2016
Distinct focal adhesion protein modules control different aspects of mechanotransduction. Journal of cell science\| PubMed ID: 28302906	2017
Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity. Journal of cell science\| PubMed ID: 28576970	2017
Mechanotransduction at the cell-matrix interface. Seminars in cell & developmental biology\| PubMed ID: 28754442	2017
Vinculin is required to maintain glomerular barrier integrity. Kidney international\| PubMed ID: 29241625	2018
GAS2-like 1 coordinates cell division through its association with end-binding proteins. Scientific reports\| PubMed ID: 30967572	2019

Christoph Ballestrem

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