Caldesmon regulates actin dynamics to influence cranial neural crest migration in Xenopus

Caldesmon (CaD) is an important actin modulator that associates with actin filaments to regulate cell morphology and motility. Although extensively studied in cultured cells, there is little functional information regarding the role of CaD in migrating cells in vivo. Here we show that nonmuscle CaD is highly expressed in both premigratory and migrating cranial neural crest cells of Xenopus embryos. Depletion of CaD with antisense morpholino oligonucleotides causes cranial neural crest cells to migrate a significantly shorter distance, prevents their segregation into distinct migratory streams, and later results in severe defects in cartilage formation. Demonstrating specificity, these effects are rescued by adding back exogenous CaD. Interestingly, CaD proteins with mutations in the Ca^(2+)-calmodulin–binding sites or ErK/Cdk1 phosphorylation sites fail to rescue the knockdown phenotypes, whereas mutation of the PAK phosphorylation site is able to rescue them. Analysis of neural crest explants reveals that CaD is required for the dynamic arrangements of actin and, thus, for cell shape changes and process formation. Taken together, these results suggest that the actin-modulating activity of CaD may underlie its critical function and is regulated by distinct signaling pathways during normal neural crest migration

Differences in protein mobility between pioneer versus follower growth cones

Navigating growth cones need to integrate, process and respond to guidance signals, requiring dynamic information transfer within and between different compartments. Studies have shown that, faced with different navigation challenges, growth cones display dynamic changes in growth kinetics and morphologies. However, it remains unknown whether these are paralleled by differences in their internal molecular dynamics. To examine whether there are protein mobility differences during guidance, we developed multiphoton fluorescence recovery after photobleaching methods to determine molecular diffusion rates in pathfinding growth cones in vivo. Actively navigating growth cones (leaders) have consistently longer recovery times than growth cones that are fasciculated and less actively navigating (followers). Pharmacological perturbations of the cytoskeleton point to actin as the primary modulator of diffusion in differently behaving growth cones. This approach provides a powerful means to quantify mobility of specific proteins in neurons in vivo and reveals that diffusion is important during axon navigation

Formins Determine the Functional Properties of Actin Filaments in Yeast

The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and func- tional properties of the actin polymer. However, the mecha- nism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filament’s ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create

The actin-myosin regulatory MRCK kinases: regulation, biological functions and associations with human cancer

The contractile actin-myosin cytoskeleton provides much of the force required for numerous cellular activities such as motility, adhesion, cytokinesis and changes in morphology. Key elements that respond to various signal pathways are the myosin II regulatory light chains (MLC), which participate in actin-myosin contraction by modulating the ATPase activity and consequent contractile force generation mediated by myosin heavy chain heads. Considerable effort has focussed on the role of MLC kinases, and yet the contributions of the myotonic dystrophy-related Cdc42-binding kinases (MRCK) proteins in MLC phosphorylation and cytoskeleton regulation have not been well characterized. In contrast to the closely related ROCK1 and ROCK2 kinases that are regulated by the RhoA and RhoC GTPases, there is relatively little information about the CDC42-regulated MRCKα, MRCKβ and MRCKγ members of the AGC (PKA, PKG and PKC) kinase family. As well as differences in upstream activation pathways, MRCK and ROCK kinases apparently differ in the way that they spatially regulate MLC phosphorylation, which ultimately affects their influence on the organization and dynamics of the actin-myosin cytoskeleton. In this review, we will summarize the MRCK protein structures, expression patterns, small molecule inhibitors, biological functions and associations with human diseases such as cancer

Actin-driven cell dynamics probed by Fourier transform light scattering

We applied the newly developed Fourier transform light scattering (FTLS) to study dynamic light scattering in single live cells, at a temporal scale of seconds to hours. The nanoscale cell fluctuations were measured with and without the active actin contribution. We found experimentally that the spatio-temporal signals rendered by FTLS reveal interesting cytoskeleton dynamics in glial cells (the predominant cell type in the nervous system). The active contribution of actin cytoskeleton was obtained by modulating its dynamic properties via Cytochalasin-D, a drug that inhibits actin polymerization/depolymerization

Focal adhesions as mechanosensors: the two-spring model

Adhesion-dependent cells actively sense the mechanical properties of their environment through mechanotransductory processes at focal adhesions, which are integrin-based contacts connecting the extracellular matrix to the cytoskeleton. Here we present first steps towards a quantitative understanding of focal adhesions as mechanosensors. It has been shown experimentally that high levels of force are related to growth of and signaling at focal adhesions. In particular, activation of the small GTPase Rho through focal adhesions leads to the formation of stress fibers. Here we discuss one way in which force might regulate the internal state of focal adhesions, namely by modulating the internal rupture dynamics of focal adhesions. A simple two-spring model shows that the stiffer the environment, the more efficient cellular force is built up at focal adhesions by molecular motors interacting with the actin filaments.Comment: Latex, 17 pages, 5 postscript figures include

Caveolin-1 Modulates Mechanotransduction Responses to Substrate Stiffness through Actin-Dependent Control of YAP

The transcriptional regulator YAP orchestrates many cellular functions, including tissue homeostasis, organ growth control, and tumorigenesis. Mechanical stimuli are a key input to YAP activity, but the mechanisms controlling this regulation remain largely uncharacterized. We show that CAV1 positively modulates the YAP mechanoresponse to substrate stiffness through actin-cytoskeleton-dependent and Hippo-kinase-independent mechanisms. RHO activity is necessary, but not sufficient, for CAV1-dependent mechanoregulation of YAP activity. Systematic quantitative interactomic studies and image-based small interfering RNA (siRNA) screens provide evidence that this actin-dependent regulation is determined by YAP interaction with the 14-3-3 protein YWHAH. Constitutive YAP activation rescued phenotypes associated with CAV1 loss, including defective extracellular matrix (ECM) remodeling. CAV1-mediated control of YAP activity was validated in vivo in a model of pancreatitis-driven acinar-to-ductal metaplasia. We propose that this CAV1-YAP mechanotransduction system controls a significant share of cell programs linked to these two pivotal regulators, with potentially broad physiological and pathological implications. Moreno-Vicente et al. report that CAV1, a key component of PM mechanosensing caveolae, mediates adaptation to ECM rigidity by modulating YAP activity through the control of actin dynamics and phosphorylation-dependent interaction of YAP with the 14-3-3-domain protein YWHAH. Cav1-dependent YAP regulation drives two pathophysiological processes: ECM remodeling and pancreatic ADM. © 2018 The Author

Transforming growth factor-beta targets Formin-like 2 for Angiopoietin-like 4 secretion during the epithelial mesenchymal transition

Epithelial to Mesenchymal transition (EMT) is a highly dynamic process that plays a crucial role in tumor progression and metastasis. While remodelling of the actin cytoskeleton is a hallmark of EMT, the responsible actin regulating factors are less well understood. Formins are involved in numerous cellular mechanisms, ranging from cytokinesis to cell adhesion and motility. The Rho-GTPase effectors of the formin family compromise the largest group of actin nucleators and are emerging as relevant pharmacological targets. A critical role of Formin-like 2 (FMNL2) in the assembly of junctional actin at newly forming cell-cell contacts in a 3D matrix has been described. This activity originates downstream of Rac1 and is in line with a physical association of FMNL2 and components of the cadherin-catenin complex. FMNL2 was further recently implicated in β1-integrin trafficking as a direct PKC target required for cancer cell invasion. Here we found that transforming growth factor-beta (TGFβ)-driven EMT leads to an upregulation of PKC resulting in the phosphorylation and activation of FMNL2 in epithelial cells. Proteomic screening for TGFβ-mediated phospho-FMNL2 binding partners identified the tumor promotor ANGPTL4 as a specific binding partner. ANGPTL4 has important roles in cancer development and progression including promoting invasion and metastasis. We found that FMNL2 and ANGPTL4 directly interact under TGF-induced EMT. Our data show that FMNL2 is a critical regulator of ANGPTL4 secretion. Secretion of ANGPTL4 is diminished upon loss of FMNL2 and its phosphorylation. We further observed that ANGPTL4 is sequestered in the Golgi apparatus colocalizing with markers of the trans-Golgi network. Live imaging of vesicle secretion from the Golgi confirmed the transient co-localization of ANGPTL4 and FMNL2. Moreover, ANGPTL4 and FMNL2 modulate cell-cell contact integrity and ANGPTL4 silenced cells fail to disassemble their underlying cell-cell contacts to execute EMT. This effect was further enhanced upon FMNL2 knockout using FMNL2 CRISPR/Cas9 cell line. However, re-introduction of ANGPTL4 restored the mesenchymal phenotype and prompted the dissolution of cell-cell adhesions. Finally, we found that cellular invasion promoted by TGFβ depends on FMNL2 and is reduced upon ANGPTL4 silencing. Taken together, our data point towards a crucial role of FMNL2 for EMT via ANGPTL4 secretion

Focal adhesions as mechanosensors: the two-spring model

Adhesion-dependent cells actively sense the mechanical properties of their environment through mechanotransductory processes at focal adhesions, which are integrin-based contacts connecting the extracellular matrix to the cytoskeleton. Here we present first steps towards a quantitative understanding of focal adhesions as mechanosensors. It has been shown experimentally that high levels of force are related to growth of and signaling at focal adhesions. In particular, activation of the small GTPase Rho through focal adhesions leads to the formation of stress fibers. Here we discuss one way in which force might regulate the internal state of focal adhesions, namely by modulating the internal rupture dynamics of focal adhesions. A simple two-spring model shows that the stiffer the environment, the more efficient cellular force is built up at focal adhesions by molecular motors interacting with the actin filaments.Comment: Latex, 17 pages, 5 postscript figures include

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin’s influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites