scholarly journals Ventral Stress Fibers Induce Plasma Membrane Deformation in Human Fibroblasts

2021 ◽  
pp. mbc.E21-03-0096
Author(s):  
Samuel J. Ghilardi ◽  
Mark S. Aronson ◽  
Allyson E. Sgro
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Human Fibroblasts ◽  
Major Axis ◽  
Ventral Side ◽  
Stress Fibers ◽  
Contractile Ring ◽  
Cellular Processes ◽  
Transmission Electron ◽  
Membrane Bending

Interactions between the actin cytoskeleton and the plasma membrane are important in many eukaryotic cellular processes. During these processes, actin structures deform the cell membrane outward by applying forces parallel to the fiber's major axis (as in migration) or they deform the membrane inward by applying forces perpendicular to the fiber's major axis (as in the contractile ring during cytokinesis). Here we describe a novel actin-membrane interaction in human dermal myofibroblasts. When labeled with a cytosolic fluorophore, the myofibroblasts developed prominent fluorescent structures on the ventral side of the cell. These structures are present in the cell membrane and colocalize with ventral actin stress fibers, suggesting that the stress fibers bend the membrane to form a “cytosolic pocket” that the fluorophores diffuse into, creating the observed structures. The existence of this pocket was confirmed by transmission electron microscopy. While dissolving the stress fibers, inhibiting fiber protein binding, or inhibiting myosin II binding of actin removed the observed pockets, decreasing cellular contractility did not remove them. Taken together, our results illustrate a novel actin-membrane bending topology where the membrane is deformed outwards rather than being pinched inwards, resembling the topological inverse of the contractile ring found in cytokinesis. [Media: see text] [Media: see text]

scholarly journals Ventral Stress Fibers Induce Plasma Membrane Deformation in Human Fibroblasts

2021 ◽  
Author(s):  
Samuel J. Ghilardi ◽  
Mark S. Aronson ◽  
Allyson E. Sgro
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Human Fibroblasts ◽  
Major Axis ◽  
Ventral Side ◽  
Stress Fibers ◽  
Cellular Processes ◽  
Transmission Electron ◽  
Fiber Protein ◽  
Membrane Bending

AbstractInteractions between the actin cytoskeleton and the plasma membrane are essential for many eukaryotic cellular processes. During these processes, actin fibers deform the cell membrane outward by applying forces parallel to the fiber’s major axis (as in migration) or they deform the membrane inward by applying forces perpendicular to the fiber’s major axis (as during cytokinesis). Here we describe a novel actin-membrane interaction in human dermal myofibroblasts. When labeled with a cytosolic fluorophore, the myofibroblasts developed prominent fluorescent structures on the ventral side of the cell. These structures are present in the cell membrane and colocalize with ventral actin stress fibers, suggesting that the fibers bend the membrane to form a “cytosolic pocket” for the fluorophores to flow into, creating the observed structures. The existence of this pocket was confirmed by transmission electron microscopy. Dissolving the stress fibers, inhibiting fiber protein binding, or inhibiting myosin II binding of actin removed the observed structures. However, decreasing cellular contractility did not remove the structures. Taken together, our results illustrate a novel actin-membrane bending topology where the membrane is deformed outwards rather than being pinched inwards, resembling the topological inverse of cytokinesis.

scholarly journals Endocytic vacuole formation by WASH-mediated endocytosis

2021 ◽  
Author(s):  
Pia Brinkert ◽  
Lena Krebs ◽  
Pilar Samperio Ventayol ◽  
Lilo Greune ◽  
Carina Bannach ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Endocytic Pathway ◽  
Vacuole Formation ◽  
Cellular Processes ◽  
Human Papillomavirus Type 16 ◽  
Systematic Perturbation ◽  
Membrane Bending ◽  
Endocytic Pathways

Endocytosis of extracellular or plasma membrane material is a fundamental process. A variety of endocytic pathways exist, several of which are barely understood in terms of mechanistic execution and biological function. Importantly, some mechanisms have been identified and characterized by following virus internalization into cells. This includes a novel endocytic pathway exploited by human papillomavirus type 16 (HPV16). However, its cellular role and mechanism of endocytic vacuole formation remain unclear. Here, HPV16 was used as a tool to examine the mechanistic execution of vesicle formation by combining systematic perturbation of cellular processes with electron and video microscopy. Our results indicate cargo uptake by uncoated, inward-budding pits facilitated by the membrane bending retromer protein SNX2. Actin polymerization-driven vesicle scission is promoted by WASH, an actin regulator typically not found at the plasma membrane. Uncovering a novel role of WASH in endocytosis, we propose to term the new pathway WASH-mediated endocytosis (WASH-ME).

scholarly journals Primary Cilia, Ciliogenesis and the Actin Cytoskeleton: A Little Less Resorption, A Little More Actin Please

2020 ◽  
Vol 8 ◽  
Author(s):  
Claire E. L. Smith ◽  
Alice V. R. Lake ◽  
Colin A. Johnson
Keyword(s):  
Cell Membrane ◽  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Primary Cilia ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Apical Surface ◽  
Apical Cell Membrane ◽  
Cellular Processes ◽  
Filament Bundles

Primary cilia are microtubule-based organelles that extend from the apical surface of most mammalian cells, forming when the basal body (derived from the mother centriole) docks at the apical cell membrane. They act as universal cellular “antennae” in vertebrates that receive and integrate mechanical and chemical signals from the extracellular environment, serving diverse roles in chemo-, mechano- and photo-sensation that control developmental signaling, cell polarity and cell proliferation. Mutations in ciliary genes cause a major group of inherited developmental disorders called ciliopathies. There are very few preventative treatments or new therapeutic interventions that modify disease progression or the long-term outlook of patients with these conditions. Recent work has identified at least four distinct but interrelated cellular processes that regulate cilia formation and maintenance, comprising the cell cycle, cellular proteostasis, signaling pathways and structural influences of the actin cytoskeleton. The actin cytoskeleton is composed of microfilaments that are formed from filamentous (F) polymers of globular G-actin subunits. Actin filaments are organized into bundles and networks, and are attached to the cell membrane, by diverse cross-linking proteins. During cell migration, actin filament bundles form either radially at the leading edge or as axial stress fibers. Early studies demonstrated that loss-of-function mutations in ciliopathy genes increased stress fiber formation and impaired ciliogenesis whereas pharmacological inhibition of actin polymerization promoted ciliogenesis. These studies suggest that polymerization of the actin cytoskeleton, F-actin branching and the formation of stress fibers all inhibit primary cilium formation, whereas depolymerization or depletion of actin enhance ciliogenesis. Here, we review the mechanistic basis for these effects on ciliogenesis, which comprise several cellular processes acting in concert at different timescales. Actin polymerization is both a physical barrier to both cilia-targeted vesicle transport and to the membrane remodeling required for ciliogenesis. In contrast, actin may cause cilia loss by localizing disassembly factors at the ciliary base, and F-actin branching may itself activate the YAP/TAZ pathway to promote cilia disassembly. The fundamental role of actin polymerization in the control of ciliogenesis may present potential new targets for disease-modifying therapeutic approaches in treating ciliopathies.

vitreal cells and specialized phagocytes in the chick embryo retina: A TEM and SEM study

1990 ◽  
Vol 48 (3) ◽  
pp. 330-331
Author(s):  
M.A. Cuadros ◽  
M.J. Martinez-Guerrero ◽  
A. Rios
Keyword(s):  
Cell Death ◽  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Chick Embryo ◽  
Basement Membrane ◽  
Sem Images ◽  
Intimate Contact ◽  
Cellular Processes ◽  
Sem Study ◽  
Free Cells

In the chick embryo retina (days 3-4 of incubation), coinciding with an increase in cell death, specialized phagocytes characterized by intense acid phosphatase activity have been described. In these preparations, all free cells in the vitreal humor (vitreal cells) were strongly labeled. Conventional TEM and SEM techniques were used to characterize them and attempt to determine their relationship with retinal phagocytes.Two types of vitreal cells were distinguished. The first are located at some distance from the basement membrane of the neuroepithelium, and are rounded, with numerous vacuoles and thin cytoplasmic prolongations. Images of exo- and or endocytosis were frequent; the cells showed a well-developed Golgi apparatus (Fig. 1) In SEM images, the cells was covered with short cellular processes (Fig. 3). Cells lying parallel to or alongside the basement membrane are elongated. The plasma membrane is frequently in intimate contact with the basement membrane. These cells have generally a large cytoplasmic expansion (Fig. 5).

scholarly journals Three pools of plasma membrane cholesterol and their relation to cholesterol homeostasis

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Akash Das ◽  
Michael S Brown ◽  
Donald D Anderson ◽  
Joseph L Goldstein ◽  
Arun Radhakrishnan
Keyword(s):  
Plasma Membrane ◽  
Regulatory Mechanism ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Mutant Form ◽  
Perfringolysin O ◽  
Cholesterol Levels ◽  
Plasma Membrane Cholesterol

When human fibroblasts take up plasma low density lipoprotein (LDL), its cholesterol is liberated in lysosomes and eventually reaches the endoplasmic reticulum (ER) where it inhibits cholesterol synthesis by blocking activation of SREBPs. This feedback protects against cholesterol overaccumulation in the plasma membrane (PM). But how does ER know whether PM is saturated with cholesterol? In this study, we define three pools of PM cholesterol: (1) a pool accessible to bind 125I-PFO*, a mutant form of bacterial Perfringolysin O, which binds cholesterol in membranes; (2) a sphingomyelin(SM)-sequestered pool that binds 125I-PFO* only after SM is destroyed by sphingomyelinase; and (3) a residual pool that does not bind 125I-PFO* even after sphingomyelinase treatment. When LDL-derived cholesterol leaves lysosomes, it expands PM's PFO-accessible pool and, after a short lag, it also increases the ER's PFO-accessible regulatory pool. This regulatory mechanism allows cells to ensure optimal cholesterol levels in PM while avoiding cholesterol overaccumulation.

Mechanism of cystine reaccumulation by cystinotic fibroblasts in vitro

1990 ◽  
Vol 10 (2) ◽  
pp. 225-229 ◽  
Author(s):  
Susan Forster ◽  
Lynne Scarlett ◽  
John B. Lloyd
Keyword(s):  
Plasma Membrane ◽  
Culture Medium ◽  
Human Fibroblasts ◽  
Competitive Inhibitor ◽  
Free Medium ◽  
Lysosome Membrane ◽  
Putative Mechanism

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

Transbilayer movement of fluorescent and spin-labeled phospholipids in the plasma membrane of human fibroblasts: a quantitative approach

1996 ◽  
Vol 109 (3) ◽  
pp. 687-698 ◽  
Author(s):  
T. Pomorski ◽  
P. Muller ◽  
B. Zimmermann ◽  
K. Burger ◽  
P.F. Devaux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Initial Velocity ◽  
Plasma Membranes ◽  
Atp Hydrolysis ◽  
Human Fibroblasts ◽  
Atp Depletion ◽  
Aminophospholipid Translocase ◽  
Order Of Magnitude ◽  
Complicating Factors ◽  
First Time

All phospholipids in the plasma membrane of eukaryotic cells are subject to a slow passive transbilayer movement. In addition, aminophospholipids are recognized by the so-called aminophospholipid translocase, and are rapidly moved from the exoplasmic to the cytoplasmic leaflet of the plasma membrane at the expense of ATP hydrolysis. Though these principal pathways of transbilayer movement of phospholipids probably apply to all eukaryotic plasma membranes, studies of the actual kinetics of phospholipid redistribution have been largely confined to non-nucleated cells (erythrocytes). Experiments on nucleated cells are complicated by endocytosis and metabolism of the lipid probes inserted into the plasma membrane. Taking these complicating factors into account, we performed a detailed kinetic study of the transbilayer movement of short-chain fluorescent (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl); NBD) and, for the first time, spin-labeled analogues of phosphatidylcholine (PC), -ethanolamine (PE), -serine (PS), and sphingomyelin (SM) in the plasma membrane of cultured human gingival fibroblasts. At 20 degrees C, the passive transbilayer diffusion of NBD analogues was very slow, and the choline-containing NBD analogues were internalized predominantly by endocytosis. Spin-labeled analogues of PC and SM showed higher passive transbilayer diffusion rates, and probably entered the cell by both passive transbilayer movement and endocytosis. In contrast, the rapid uptake of NBD- and spin-labeled aminophospholipid analogues could be mainly ascribed to the action of the aminophospholipid translocase, since it was inhibited by ATP depletion and N-ethylmaleimide pretreatment. The initial velocity of NBD-aminophospholipid translocation was eight to ten times slower than that of the corresponding spin-labeled lipid, and the half-times of redistribution of NBD-PS and spin-labeled PS were 7.2 and 3.6 minutes, respectively. Our data indicate that in human fibroblasts the initial velocity of aminophospholipid translocation is at least one order of magnitude higher than that in human erythrocytes, which should be sufficient to maintain the phospholipid asymmetry in the plasma membrane.

Time-Course of Changes in the Plasma–Membrane Lipid Bilayer and Cellular Ultrastructure Induced by Tumour Necrosis Factor-α in K 562 and Daudi Cells

1995 ◽  
Vol 23 (4) ◽  
pp. 254-263 ◽  
Author(s):  
M Marutaka ◽  
H Iwagaki ◽  
K Mizukawa ◽  
N Tanaka ◽  
K Orita
Keyword(s):  
Plasma Membrane ◽  
Cell Membrane ◽  
Membrane Fluidity ◽  
Time Course ◽  
Membrane Lipid ◽  
Plasma Membrane Lipid ◽  
Membrane Lipid Bilayer ◽  
Cell Membrane Fluidity ◽  
K 562 Cells ◽  
Factor Α

The time-course of changes in the plasma-membrane lipid bilayer induced by tumour necrosis factor-α (TNF) were investigated in cultured cells using spin-label electron-spin-resonance techniques. Treatment of K 562 cells, a human chronic myelocytic leukaemia cell line, in suspension culture with TNF for up to 6 h caused an initial increase in cell-membrane fluidity, which returned to the control level after 12 h of treatment. After 24 h of treatment, the cell-membrane fluidity had decreased and this decrease was maintained after 48 h of treatment. In Daudi cells, a human malignant lymphoma cell line, TNF, did not induce any changes in cell-membrane fluidity, indicating that the effect of TNF on membrane structure is cell-specific. The early and transient change in membrane fluidity in K 562 cells is probably related to signal generation, while the later, persistent change may reflect the phenotype of TNF-treated cells, in particular, changes in the plasma membrane-cytoplasmic complex. Histochemical electron microscopic studies indicated that the membrane fluidity changes induced by TNF have an ultrastructural correlate.

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