scholarly journals Dynamic actin crosslinking governs the cytoplasm’s transition to fluid-like behavior

2019 ◽  
Author(s):  
Loïc Chaubet ◽  
Abdullah R. Chaudhary ◽  
Hossein K. Heris ◽  
Allen J. Ehrlicher ◽  
Adam G. Hendricks
Keyword(s):  
Viscoelastic Properties ◽  
Myosin Ii ◽  
Optical Trap ◽  
Living Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Long Timescales

AbstractCells precisely control their mechanical properties to organize and differentiate into tissues. The architecture and connectivity of cytoskeletal filaments changes in response to mechanical and biochemical cues, allowing the cell to rapidly tune its mechanics from highly-crosslinked, elastic networks to weakly-crosslinked viscous networks. While the role of actin crosslinking in controlling actin network mechanics is well-characterized in purified actin networks, its mechanical role in the cytoplasm of living cells remains unknown. Here, we probe the frequency-dependent intracellular viscoelastic properties of living cells using multifrequency excitation and in situ optical trap calibration. At long timescales in the intracellular environment, we observe that the cytoskeleton becomes fluid-like. The mechanics are well-captured by a model in which actin filaments are dynamically connected by a single dominant crosslinker. A disease-causing point mutation (K255E) of the actin crosslinker α-actinin 4 (ACTN4) causes its binding kinetics to be insensitive to tension. Under normal conditions, the viscoelastic properties of wild type (WT) and K255E+/- cells are similar. However, when tension is reduced through myosin II inhibition, WT cells relax 3x faster to the fluid-like regime while K255E+/- cells are not affected. These results indicate that dynamic actin crosslinking enables the cytoplasm to flow at long timescales.

scholarly journals Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly

2013 ◽  
Vol 200 (5) ◽  
pp. 619-633 ◽  
Author(s):  
Elena Ingerman ◽  
Jennifer Ying Hsiao ◽  
R. Dyche Mullins
Keyword(s):  
Atp Hydrolysis ◽  
Leading Edge ◽  
S2 Cells ◽  
Actin Network ◽  
Wild Type ◽  
Actin Networks ◽  
Dendritic Networks ◽  
A Cell

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

scholarly journals Transport and localization of exogenous myelin basic protein mRNA microinjected into oligodendrocytes.

1993 ◽  
Vol 123 (2) ◽  
pp. 431-441 ◽  
Author(s):  
K Ainger ◽  
D Avossa ◽  
F Morgan ◽  
S J Hill ◽  
C Barry ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Living Cells ◽  
Branch Points ◽  
Rna Granules ◽  
Intracellular Movement ◽  
Directional Movement ◽  
Ionic Detergent ◽  
Mrna Granules

We have studied transport and localization of MBP mRNA in oligodendrocytes in culture by microinjecting labeled mRNA into living cells and analyzing the intracellular distribution of the injected RNA by confocal microscopy. Injected mRNA initially appears dispersed in the perikaryon. Within minutes, the RNA forms granules which, in the case of MBP mRNA, are transported down the processes to the periphery of the cell where the distribution again becomes dispersed. In situ hybridization shows that endogenous MBP mRNA in oligodendrocytes also appears as granules in the perikaryon and processes and dispersed in the peripheral membranes. The granules are not released by extraction with non-ionic detergent, indicating that they are associated with the cytoskeletal matrix. Three dimensional visualization indicates that MBP mRNA granules are often aligned in tracks along microtubules traversing the cytoplasm and processes. Several distinct patterns of granule movement are observed. Granules in the processes undergo sustained directional movement with a velocity of approximately 0.2 micron/s. Granules at branch points undergo oscillatory motion with a mean displacement of 0.1 micron/s. Granules in the periphery of the cell circulate randomly with a mean displacement of approximately 1 micron/s. The results are discussed in terms of a multi-step pathway for transport and localization of MBP mRNA in oligodendrocytes. This work represents the first characterization of intracellular movement of mRNA in living cells, and the first description of the role of RNA granules in transport and localization of mRNA in cells.

Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

2014 ◽  
Vol 306 (6) ◽  
pp. C607-C620 ◽  
Author(s):  
Hiroaki Hirata ◽  
Hitoshi Tatsumi ◽  
Chwee Teck Lim ◽  
Masahiro Sokabe
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Adhesions ◽  
Living Cells ◽  
Live Cells ◽  
Mechanical Forces ◽  
Actin Network ◽  
Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice

1999 ◽  
Vol 277 (2) ◽  
pp. F298-F302 ◽  
Author(s):  
Tong Wang ◽  
Chao-Ling Yang ◽  
Thecla Abbiati ◽  
Patrick J. Schultheis ◽  
Gary E. Shull ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Proximal Tubule ◽  
Apical Membrane ◽  
Significant Inhibition ◽  
Fluid Absorption ◽  
Wild Type ◽  
Significant Component ◽  
Nhe Isoforms

NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3−/−mouse with greatly reduced proximal tubule[Formula: see text] absorption compared with NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282–285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule [Formula: see text] reabsorption in NHE3−/− mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of[Formula: see text] ( J HCO3) and fluid absorption ( J v) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 μM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J HCO3 and J v in NHE3+/+ mice but failed to inhibit J HCO3 or J v in NHE3−/− mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 μM bafilomycin caused a similar absolute decrement in J HCO3 in wild-type and NHE3 null mice, indicating equivalent rates of[Formula: see text] absorption mediated by H+-ATPase. Addition of 10 μM Sch-28080 did not reduce J HCO3 in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating [Formula: see text] reabsorption in the proximal tubule. A significant component of[Formula: see text] reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

scholarly journals Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

2017 ◽  
Author(s):  
Alyson S. Smith ◽  
Roberta B. Nowak ◽  
Sitong Zhou ◽  
Michael Giannetto ◽  
David S. Gokhin ◽  
...  
Keyword(s):  
Motor Activity ◽  
Myosin Ii ◽  
Control Cell ◽  
Cell Types ◽  
Membrane Skeleton ◽  
Membrane Curvature ◽  
General Function ◽  
Actin Network ◽  
Actin Networks ◽  
Cell Shapes

AbstractThe biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylatedin vivoin RBCs, indicating active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disc cell shape. As structures similar to the RBC membrane skeleton are conserved in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.Significance statementThe biconcave disc shape and deformability of the mammalian RBC is vital to its circulatory function, relying upon a 2D viscoelastic spectrin-F-actin network attached to the membrane. A role for myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has never been tested. We show that NMIIA forms phosphorylated bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity is required for interactions with the spectrin-F-actin network, and regulates RBC biconcave shape and deformability. These results provide a novel mechanism for actomyosin force generation at the plasma membrane, and may be applicable to other cell types such as neurons and polarized epithelial cells with a spectrin-F-actin-based membrane skeleton.

scholarly journals On the Role of Myosin-II in Cytokinesis: Division ofDictyostelium Cells under Adhesive and Nonadhesive Conditions

1997 ◽  
Vol 8 (12) ◽  
pp. 2617-2629 ◽  
Author(s):  
Ji-Hong Zang ◽  
Guy Cavet ◽  
James H. Sabry ◽  
Peter Wagner ◽  
Sheri L. Moores ◽  
...  
Keyword(s):  
Cell Division ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Ii ◽  
Hydrophobic Surface ◽  
Wild Type ◽  
Daughter Cells ◽  
Adhesive Surface ◽  
Spatial And Temporal Changes

We have investigated the role of myosin in cytokinesis inDictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (ΔBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing ΔBLCBS-myosin were previously shown to divide in suspension ( Uyeda et al., 1996 ). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, ΔBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a ΔBLCBS-myosin is similar to that in wild-type cells.

scholarly journals Acid Sphingomyelinase Deficiency Increases Susceptibility to Fatal Alphavirus Encephalomyelitis

2006 ◽  
Vol 80 (22) ◽  
pp. 10989-10999 ◽  
Author(s):  
Ching G. Ng ◽  
Diane E. Griffin
Keyword(s):  
Sindbis Virus ◽  
Acid Sphingomyelinase ◽  
Wild Type ◽  
Fatal Disease ◽  
Necrosis Factor Alpha ◽  
Enveloped Virus ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Sindbis virus (SV), an enveloped virus with a single-stranded, plus-sense RNA genome, is the prototype alphavirus in the Togaviridae family. In mice, SV infects neurons and can cause apoptosis of immature neurons. Sphingomyelin (SM) is the most prevalent cellular sphingolipid, is particularly abundant in the nervous systems of mammals, and is required for alphavirus fusion and entry. The level of SM is tightly regulated by sphingomyelinases. A defect in acid sphingomyelinase (ASMase) results in SM storage and subsequent intracellular accumulation of SM. To better understand the role of the SM pathway in SV pathogenesis, we have characterized SV infection of transgenic mice deficient in the ASMase gene. ASMase knockout (ASM-KO) mice were more susceptible to SV infection than wild-type (WT) or heterozygous (Het) animals. Titers of SV were higher in the brains of ASM-KO mice than in the brains of WT mice. More SV RNA was detected by in situ hybridization, more SV protein was detected by immunohistochemistry, and more terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive cells were present in the cortex and hippocampus of ASM-KO mice than in those of WT or Het mice. Interleukin-6 (IL-6), but not IL-1β or tumor necrosis factor alpha, was elevated in infected ASM-KO mice compared to levels in WT or Het mice, but studies with IL-6-KO mice and recombinant SV expressing IL-6 showed no role for IL-6 in fatal disease. Together these data indicate that the increase in susceptibility of ASM-KO mice to SV infection was the result of more-rapid replication and spread of SV in the nervous system and increased neuronal death.

scholarly journals Concerted actions of distinct nonmuscle myosin II isoforms drive intracellular membrane remodeling in live animals

2017 ◽  
Vol 216 (7) ◽  
pp. 1925-1936 ◽  
Author(s):  
Oleg Milberg ◽  
Akiko Shitara ◽  
Seham Ebrahim ◽  
Andrius Masedunskas ◽  
Muhibullah Tora ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Myosin Ii ◽  
Membrane Remodeling ◽  
Nonmuscle Myosin ◽  
Nonmuscle Myosin Ii ◽  
Mammalian Tissues ◽  
Actomyosin Cytoskeleton

Membrane remodeling plays a fundamental role during a variety of biological events. However, the dynamics and the molecular mechanisms regulating this process within cells in mammalian tissues in situ remain largely unknown. In this study, we use intravital subcellular microscopy in live mice to study the role of the actomyosin cytoskeleton in driving the remodeling of membranes of large secretory granules, which are integrated into the plasma membrane during regulated exocytosis. We show that two isoforms of nonmuscle myosin II, NMIIA and NMIIB, control distinct steps of the integration process. Furthermore, we find that F-actin is not essential for the recruitment of NMII to the secretory granules but plays a key role in the assembly and activation of NMII into contractile filaments. Our data support a dual role for the actomyosin cytoskeleton in providing the mechanical forces required to remodel the lipid bilayer and serving as a scaffold to recruit key regulatory molecules.

scholarly journals Attenuated Late-PhaseArcTranscription in the Dentate Gyrus of Mice LackingEgr3

2017 ◽  
Vol 2017 ◽  
pp. 1-6 ◽  
Author(s):  
Amanda Maple ◽  
Rachel E. Lackie ◽  
Diana I. Elizalde ◽  
Stephanie L. Grella ◽  
Chelsey C. Damphousse ◽  
...  
Keyword(s):  
Dentate Gyrus ◽  
Time Course ◽  
Wild Type ◽  
Short Term ◽  
Unique Role ◽  
Late Protein ◽  
Dependent Learning

The dentate gyrus (DG) engages in sustainedArctranscription for at least 8 hours following behavioral induction, and this time course may be functionally coupled to the unique role of the DG in hippocampus-dependent learning and memory. The factors that regulate long-term DGArcexpression, however, remain poorly understood. Animals lackingEgr3show lessArcexpression following convulsive stimulation, but the effect ofEgr3ablation on behaviorally inducedArcremains unknown. To address this,Egr3−/−and wild-type (WT) mice explored novel spatial environments and were sacrificed either immediately or after 5, 60, 240, or 480 minutes, andArcexpression was quantified by fluorescence in situ hybridization. Although short-term (i.e., within 60 min)Arcexpression was equivalent across genotypes, DGArcexpression was selectively reduced at 240 and 480 minutes in mice lackingEgr3. These data demonstrate the involvement ofEgr3in regulating the late protein-dependent phase ofArcexpression in the DG.

scholarly journals Control of nuclear centration in the C. elegans zygote by receptor-independent Gα signaling and myosin II

2007 ◽  
Vol 178 (7) ◽  
pp. 1177-1191 ◽  
Author(s):  
Morgan B. Goulding ◽  
Julie C. Canman ◽  
Eric N. Senning ◽  
Andrew H. Marcus ◽  
Bruce Bowerman
Keyword(s):  
Mitotic Spindle ◽  
Myosin Ii ◽  
Nonmuscle Myosin ◽  
Wild Type ◽  
Spindle Positioning ◽  
C Elegans ◽  
Nonmuscle Myosin Ii ◽  
Pulling Forces ◽  
Actomyosin Contraction

Mitotic spindle positioning in the Caenorhabditis elegans zygote involves microtubule-dependent pulling forces applied to centrosomes. In this study, we investigate the role of actomyosin in centration, the movement of the nucleus–centrosome complex (NCC) to the cell center. We find that the rate of wild-type centration depends equally on the nonmuscle myosin II NMY-2 and the Gα proteins GOA-1/GPA-16. In centration- defective let-99(−) mutant zygotes, GOA-1/GPA-16 and NMY-2 act abnormally to oppose centration. This suggests that LET-99 determines the direction of a force on the NCC that is promoted by Gα signaling and actomyosin. During wild-type centration, NMY-2–GFP aggregates anterior to the NCC tend to move further anterior, suggesting that actomyosin contraction could pull the NCC. In GOA-1/GPA-16–depleted zygotes, NMY-2 aggregate displacement is reduced and largely randomized, whereas in a let-99(−) mutant, NMY-2 aggregates tend to make large posterior displacements. These results suggest that Gα signaling and LET-99 control centration by regulating polarized actomyosin contraction.

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