Myosin II-Mediated Focal Adhesion Maturation Is Tension Insensitive

Myosin II motors drive changes in focal adhesion morphology and composition in a “maturation process” that is crucial for regulating adhesion dynamics and signaling guiding cell adhesion, migration and fate. The underlying mechanisms of maturation, however, have been obscured by the intermingled effects of myosin II on lamellar actin architecture, dynamics and force transmission. Here, we show that focal adhesion growth rate stays constant even when cellular tension is reduced by 75%. Focal adhesion growth halts only when myosin stresses are sufficiently low to impair actin retrograde flow. Focal adhesion lifetime is reduced at low levels of cellular tension, but adhesion stability can be rescued at low levels of force by over-expression of α-actinin or constitutively active Dia1. Our work identifies a minimal myosin activity threshold that is necessary to drive lamellar actin retrograde flow is sufficient to permit focal adhesion elongation. Above this nominal threshold, myosin-mediated actin organization and dynamics regulate focal adhesion growth and stability in a force-insensitive fashion.</p

Alumni Voices

Session was facilitated by Dr. Lawrence Burnley and moderated by Dr. Daria Graham ’92 ’01 ’18, associate vice president for student affairs and dean of students at California State University, San Bernardino. Panelists included Angela Heath ’78 ’80; Darius Beckham ’19; Lisa Rich-Milan ’85; and Dr. Marcus Smith ’08 ’10. These proceedings are available free for download but also available for purchase in print for $6 plus tax and shipping.https://ecommons.udayton.edu/global_voices_4/1009/thumbnail.jp

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

Myosin II-mediated focal adhesion maturation is tension insensitive.

Myosin II motors drive changes in focal adhesion morphology and composition in a "maturation process" that is crucial for regulating adhesion dynamics and signaling guiding cell adhesion, migration and fate. The underlying mechanisms of maturation, however, have been obscured by the intermingled effects of myosin II on lamellar actin architecture, dynamics and force transmission. Here, we show that focal adhesion growth rate stays constant even when cellular tension is reduced by 75%. Focal adhesion growth halts only when myosin stresses are sufficiently low to impair actin retrograde flow. Focal adhesion lifetime is reduced at low levels of cellular tension, but adhesion stability can be rescued at low levels of force by over-expression of α-actinin or constitutively active Dia1. Our work identifies a minimal myosin activity threshold that is necessary to drive lamellar actin retrograde flow is sufficient to permit focal adhesion elongation. Above this nominal threshold, myosin-mediated actin organization and dynamics regulate focal adhesion growth and stability in a force-insensitive fashion

Arp2/3 inhibition induces amoeboid-like protrusions in MCF10A epithelial cells by reduced cytoskeletal-membrane coupling and focal adhesion assembly.

Here we demonstrate that Arp2/3 regulates a transition between mesenchymal and amoeboid protrusions in MCF10A epithelial cells. Using genetic and pharmacological means, we first show Arp2/3 inhibition impairs directed cell migration. Arp2/3 inhibition results in a dramatically impaired cell adhesion, causing deficient cell attachment and spreading to ECM as well as an 8-fold decrease in nascent adhesion assembly at the leading edge. While Arp2/3 does not play a significant role in myosin-dependent adhesion growth, mature focal adhesions undergo large scale movements against the ECM suggesting reduced coupling to the ECM. Cell edge protrusions occur at similar rates when Arp2/3 is inhibited but their morphology is dramatically altered. Persistent lamellipodia are abrogated and we observe a markedly increased incidence of blebbing and unstable pseuodopods. Micropipette-aspiration assays indicate that Arp2/3-inhibited cells have a weak coupling between the cell cortex and the plasma membrane, and suggest a potential mechanism for increased pseudopod and bleb formation. Pseudopods are not sensitive to reduced in formin or myosin II activity. Collectively, these results indicate that Arp2/3 is not necessary for rapid protrusion of the cell edge but plays a crucial role in assembling focal adhesions required for its stabilization

Arp2/3 Inhibition Induces Amoeboid-Like Protrusions in MCF10A Epithelial Cells by Reduced Cytoskeletal-Membrane Coupling and Focal Adhesion Assembly

Here we demonstrate that Arp2/3 regulates a transition between mesenchymal and amoeboid protrusions in MCF10A epithelial cells. Using genetic and pharmacological means, we first show Arp2/3 inhibition impairs directed cell migration. Arp2/3 inhibition results in a dramatically impaired cell adhesion, causing deficient cell attachment and spreading to ECM as well as an 8-fold decrease in nascent adhesion assembly at the leading edge. While Arp2/3 does not play a significant role in myosin-dependent adhesion growth, mature focal adhesions undergo large scale movements against the ECM suggesting reduced coupling to the ECM. Cell edge protrusions occur at similar rates when Arp2/3 is inhibited but their morphology is dramatically altered. Persistent lamellipodia are abrogated and we observe a markedly increased incidence of blebbing and unstable pseuodopods. Micropipette-aspiration assays indicate that Arp2/3-inhibited cells have a weak coupling between the cell cortex and the plasma membrane, and suggest a potential mechanism for increased pseudopod and bleb formation. Pseudopods are not sensitive to reduced in formin or myosin II activity. Collectively, these results indicate that Arp2/3 is not necessary for rapid protrusion of the cell edge but plays a crucial role in assembling focal adhesions required for its stabilization.</p

Focal Adhesion Maturation under Treatment with ROCK inhibitor Y-27632.

<p>(a) Time-lapse images of GFP-actin and mApple-paxillin in U2OS cell treated with 2 µM Y-27632, with images of traction stress magnitude. Time is in min:sec. (b) Focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion indicated in (a) as a function of time. Focal adhesion assembly characteristics in varied Y-27632 concentrations: (c) FA elongation rate, (d) FA loading rate, (e) FA disassembly rate, (f) FA lifetime under indicated conditions (n = 10 FA for each condition for FA elongation, loading rate, disassembly rate, and lifetime). (g) Lamellar actin retrograde flow speeds in varied conditions (n = 2 cells per condition, 5 to 7 regions per cell). (h) Box plot of peak traction stress reached during focal adhesion lifetimes in cells treated with Y-27632. “Stable FA” indicates FA that had lifetime greater than 30 minutes, while “Unstable FA” indicates FA with lifetime less than 30 minutes.</p

Focal Adhesion Morphology and Traction Forces under Treatment with ROCK inhibitor Y-27632.

<p>(a) Images of F-actin visualized by fluorescent phalloidin (top row) and paxillin immunofluorescence (middle row) in U2OS cells treated with the indicated concentrations of the rho-kinase inhibitor, Y-27632. (b) Box plots of FA lengths in U2OS cells as a function of Y-27632 concentration (n = 101 (WT), 78 (1 µM), 134 (2 µM), 117 (5 µM), 124 (10 µM) FAs). (c) Representative GFP-actin images (top row) and traction stress maps (bottom row) and for U2OS cells treated with 2, 5 and 10 µM Y-27632. (d) Plot of the total traction force exerted by U2OS cells as a function of Y-27632 concentration (n = 23 (WT), 13 (1 µM), 8 (2 µM), 14 (5 µM), 6 (10 µM) cells).</p

Arp2/3 inhibited cells show defects in spreading.

<p>(<b>A</b>) Time series of untreated (wild type) cells or cells treated with 50 µM of CK-869; time indicated in minutes. Panels to the right show representative graph of spread area versus time for a cell in the cells presented in A. Scale bar = 10 µm (<b>B</b>) Rates of cell spreading in wildtype MCF10A cells or cells treated with 50 µM CK-869. Rates of spreading were calculated from the slopes of kymographs of the cell edge (n = 22 cells for wild type and 21 cells for CK-869). (<b>C</b>) Fraction of cells that retracted after reaching their maximum spread area. The ratio of the full spread area (FSA) to the smallest spread area (SSA) was calculated and if FSA/SSA >2, the cell was included as a retraction (n = 53 untreated cells, n = 64 for 50 µM control compound (CK-312) and n = 35 and 27 cells for 25 and 50 µM CK-869, respectively). (<b>D</b>) Bar graph of the time in hours to reach maximum spread area for untreated MCF10A cells or cells treated with 50 µM CK-869. NS, not significant; *, P<0.05 with respect to WT or control.</p