Tropomyosin Tm5NM1 Spatially Restricts Src Kinase Activity through Perturbation of Rab11 Vesicle Trafficking

Abstract

dIn order for cells to stop moving, they must synchronously stabilize actin filaments and their associated focal adhesions. How these two structures are coordinated in time and space is not known. We show here that the actin association protein Tm5NM1, which induces stable actin filaments, concurrently suppresses the trafficking of focal-adhesion-regulatory molecules. Using combinations of fluorescent biosensors and fluorescence recovery after photobleaching (FRAP), we demonstrate that Tm5NM1 reduces the level of delivery of Src kinase to focal adhesions, resulting in reduced phosphorylation of adhesion-resident Src substrates. Live imaging of Rab11-positive recycling endosomes that carry Src to focal adhesions reveals disruption of this pathway. We propose that tropomyosin synchronizes adhesion dynamics with the cytoskeleton by regulating actin-dependent trafficking of essential focal-adhesion molecules. Cell migration is essential for normal embryonic development, wound healing, and immune response, and migration dysregulation underpins an array of pathological conditions. Much focus is given to the initiation and speed of migration; however, of equal importance are the mechanisms by which migration is arrested. Integrin-based focal adhesions, which are bound on the external surface to the extracellular matrix and internally to the actin cytoskeleton, are key structural elements of the machinery that drives cell migration (1). While actin filament stabilization inhibits cell migration (2-6), little is known about how the associated focal adhesions are synchronously stabilized along with actin filaments. This is critical, because events that trigger focal adhesion disassembly correspondingly break the adhesion-cytoskeleton linkage (7-9), facilitating new cycles of membrane protrusion and forward movement (10). Thus, in order to stop migrating, cells must have mechanisms to synchronize actin and adhesion stability.The tropomyosins are a multi-isoform family of actin-binding proteins that display spatially and temporally restricted association with discrete actin filament populations (11). Dimers of tropomyosin associate head to tail, forming a coiled polymer that lies in the major groove of the actin filament. The association of discrete tropomyosin isoforms is thought to regulate the dynamic state of actin filaments by affecting the association of other actinregulatory proteins (12). Thus, cells with altered tropomyosin expression profiles display differential actin filament stability (2, 4, 13). Moreover, tropomyosin isoform expression is correlated with different focal-adhesion morphology (4, 14) and isoform-specific effects on cell migration (2, 4, 14-16). The tropomyosin isoform Tm5NM1 is ubiquitously expressed (17), and elevated expression inhibits both 2-dimensional (2D) (2, 4) and 3D (16) cell migration. In line with these migration effects, Tm5NM1 induces actin filament stability (13) coupled with highly stabilized focal adhesions (4).Tm5NM1-mediated actin filament stabilization is accompanied by reduce...

Rab11 collaborates E‐cadherin to promote collective cell migration and indicates a poor prognosis in colorectal carcinoma

“…Rab11 has been shown to function in the movement of recycled endosomes to the membrane and regulates epithelial cell polarity [32,33]. It has also been reported to participate in cellular functions and cancerous metastasis [29,[34][35][36][37][38][39]. Our previous work further demonstrated that Rab11 can regulate E-cadherin expression during cell transformation in colon cancer [19].…”

Small molecule targeting of the actin associating protein tropomyosin Tpm3.1 increases neuroblastoma cell response to inhibition of Rac‐mediated multicellular invasion

“…Proteins were extracted as previously described (Bach et al, ). Protein concentrations were equalised following quantification with the Biocinchoninic acid (BCA) Protein Assay Kit (Pierce Biotechnology).…”

“…Their major function is to regulate the dynamics of the associated actin filament, via regulating access of different myosin motors (Bryce et al, ; Clayton et al, ; Manstein, & Mulvihilll, ; Schevzov et al, ). Tpm3.1 stabilises actin filaments, through myosin II recruitment (Bryce et al, ; Clayton, Pollard, Murray, & Lord, 2015; Gateva et al, ; Schevzov et al, ) and this stabilising function underpins the multiplicity of roles that have been ascribed to this ubiquitously expressed protein (Bach et al, 2009; ; Bryce et al, ; Caldwell et al, ; Clayton et al, ; Lees, Bach, Bradbury, Lees et al, , 2013; Lim et al, ; Schevzov et al, ; Vlahovich et al, ). Elevated Tpm3.1 expression inhibits both 2‐dimensional (2D) and 3D single cell migration (Bach et al, 2009; ; Bryce et al, ; Lees, Bach, Bradbury, Lees et al, ).…”

“…Tpm3.1 stabilises actin filaments, through myosin II recruitment (Bryce et al, ; Clayton, Pollard, Murray, & Lord, 2015; Gateva et al, ; Schevzov et al, ) and this stabilising function underpins the multiplicity of roles that have been ascribed to this ubiquitously expressed protein (Bach et al, 2009; ; Bryce et al, ; Caldwell et al, ; Clayton et al, ; Lees, Bach, Bradbury, Lees et al, , 2013; Lim et al, ; Schevzov et al, ; Vlahovich et al, ). Elevated Tpm3.1 expression inhibits both 2‐dimensional (2D) and 3D single cell migration (Bach et al, 2009; ; Bryce et al, ; Lees, Bach, Bradbury, Lees et al, ). Conversely, the loss of Tpm3.1 expression, either through genetic deletion or siRNA‐mediated depletion, is inversely related to Rac GTPase activity and thus stimulates Rac‐dependent, elongated mesenchymal invasion in single invasive cells (Lees, Bach, Bradbury, et al, 2011; Lees et al, ).…”

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

“…Moreover, RCP/Rab11-driven recycling of ␣5␤1-integrin controls the activation of Rac and RhoA in different cell lines (A2780, MDA-MB-231 and HT1080 cells) (Jacquemet et al, 2013). The recycling of ␤1-integrin is known to be regulated by Arf6 and Rab11 (Eva et al, 2010(Eva et al, , 2012Powelka et al, 2004) and Rab11 was also implied in the targeted delivery of Src tyrosine kinases to FAs (Bach et al, 2014) where Src contributes to the phosphorylation of FAK (Canel et al, 2013). In addition, ␤1-integrin recruitment can lead to an activation of Rac1 in HeLa cells (O'Toole et al, 2011).…”