The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.
© 2018, Fenix et al.
Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.
© 2017 Lehtimäki et al.
Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.
© 2017 Erdogan et al.
Class III myosins (MYO3A and MYO3B) are proposed to function as transporters as well as length and ultrastructure regulators within stable actin-based protrusions such as stereocilia and calycal processes. MYO3A differs from MYO3B in that it contains an extended tail domain with an additional actin-binding motif. We examined how the properties of the motor and tail domains of human class III myosins impact their ability to enhance the formation and elongation of actin protrusions. Direct examination of the motor and enzymatic properties of human MYO3A and MYO3B revealed that MYO3A is a 2-fold faster motor with enhanced ATPase activity and actin affinity. A chimera in which the MYO3A tail was fused to the MYO3B motor demonstrated that motor activity correlates with formation and elongation of actin protrusions. We demonstrate that removal of individual exons (30-34) in the MYO3A tail does not prevent filopodia tip localization but abolishes the ability to enhance actin protrusion formation and elongation in COS7 cells. Interestingly, our results demonstrate that MYO3A slows filopodia dynamics and enhances filopodia lifetime in COS7 cells. We also demonstrate that MYO3A is more efficient than MYO3B at increasing formation and elongation of stable microvilli on the surface of cultured epithelial cells. We propose that the unique features of MYO3A, enhanced motor activity, and an extended tail with tail actin-binding motif, allow it to play an important role in stable actin protrusion length and ultrastructure maintenance.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
The final steps of cell division are tightly coordinated in space and time, but whether mechanisms exist to couple the actin and microtubule (MT) cytoskeletons during anaphase and cytokinesis (C phase) is largely unknown. During anaphase, MTs are incorporated into an anti-parallel array termed the spindle midzone (midzone MTs), whereas F-actin and non-muscle myosin II, together with other factors, organize into the cleavage furrow . Previous studies in somatic cells have shown that midzone MTs become highly stable after furrows have begun ingression , indicating that furrow-to-MT communication may occur. Midzone formation is also inhibited in fly spermatocytes that fail to form a cleavage furrow  and during monopolar cytokinesis when myosin contractility is blocked by blebbistatin . We show here that midzone MT stabilization is dependent on actomyosin contraction, suggesting that there is active coordination between furrow ingression and microtubule dynamics. Midzone microtubule stabilization also depends on the kinase activity of Aurora B, the catalytic subunit of the chromosomal passenger complex (CPC), uncovering a feedback mechanism that couples furrowing with microtubule dynamics. We further show that the CPC scaffolding protein INCENP (inner centromere protein) binds actin, an interaction that is important for cytokinesis and for midzone MT stabilization following furrow ingression. Stabilization of midzone MTs with low amounts of Taxol rescues cytokinesis in INCENP actin-binding mutant-expressing cells. Collectively, our work demonstrates that the actin and microtubule cytoskeletons are coordinated during cytokinesis and suggests that the CPC is integral for coupling furrow ingression with midzone microtubule stabilization.
Copyright © 2016 Elsevier Ltd. All rights reserved.
ROCK activity increases due to ECM rigidity in the tumor microenvironment and promotes a malignant phenotype via actomyosin contractility. Invasive migration is facilitated by actin-rich adhesive protrusions known as invadopodia that degrade the ECM. Invadopodia activity is dependent on matrix rigidity and contractile forces suggesting that mechanical factors may regulate these subcellular structures through ROCK-dependent actomyosin contractility. However, emerging evidence indicates that the ROCK1 and ROCK2 isoforms perform different functions in cells suggesting that alternative mechanisms may potentially regulate rigidity-dependent invadopodia activity. In this study, we found that matrix rigidity drives ROCK signaling in cancer cells but that ROCK1 and ROCK2 differentially regulate invadopodia activity through separate signaling pathways via contractile (NM II) and non-contractile (LIMK) mechanisms. These data suggest that the mechanical rigidity of the tumor microenvironment may drive ROCK signaling through distinct pathways to enhance the invasive migration required for cancer progression and metastasis.
Copyright © 2016 Elsevier Ltd. All rights reserved.
In vertebrate epithelia, p120-catenin (hereafter referred to as p120; also known as CTNND1) mediates E-cadherin stability and suppression of RhoA. Genetic ablation of p120 in various epithelial tissues typically causes striking alterations in tissue function and morphology. Although these effects could very well involve p120's activity towards Rho, ascertaining the impact of this relationship has been complicated by the fact that p120 is also required for cell-cell adhesion. Here, we have molecularly uncoupled p120's cadherin-stabilizing and RhoA-suppressing activites. Unexpectedly, removing p120's Rho-suppressing activity dramatically disrupted the integrity of the apical surface, irrespective of E-cadherin stability. The physical defect was tracked to excessive actomyosin contractility along the vertical axis of lateral membranes. Thus, we suggest that p120's distinct activities towards E-cadherin and Rho are molecularly and functionally coupled and this, in turn, enables the maintenance of cell shape in the larger context of an epithelial monolayer. Importantly, local suppression of contractility by cadherin-bound p120 appears to go beyond regulating cell shape, as loss of this activity also leads to major defects in epithelial lumenogenesis.
© 2016. Published by The Company of Biologists Ltd.
A migrating cell must establish front-to-back polarity in order to move. In this issue, Juanes-Garcia et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201407059) report that a short serine-rich motif in nonmuscle myosin IIB is required to establish the cell's rear. This motif represents a new paradigm for what determines directional cell migration.
© 2015 Fenix and Burnette.
Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.