scholarly journals Microtubules support a disk-like septin arrangement at the plasma membrane of mammalian cells

2011 ◽  
Vol 22 (23) ◽  
pp. 4588-4601 ◽  
Author(s):  
Mikael E. Sellin ◽  
Per Holmfeldt ◽  
Sonja Stenmark ◽  
Martin Gullberg
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Cell Model ◽  
Cell Types ◽  
Higher Order ◽  
Model Systems ◽  
Animal Cells ◽  
Endogenous Gene ◽  
Binding Domains ◽  
Substrate Adhesion

Septin family proteins oligomerize through guanosine 5′-triphosphate–binding domains into core heteromers, which in turn polymerize at the cleavage furrow of dividing fungal and animal cells. Septin assemblies during the interphase of animal cells remain poorly defined and are the topic of this report. In this study, we developed protocols for visualization of authentic higher-order assemblies using tagged septins to effectively replace the endogenous gene product within septin core heteromers in human cells. Our analysis revealed that septins assemble into microtubule-supported, disk-like structures at the plasma membrane. In the absence of cell substrate adhesion, this is the predominant higher-order arrangement in interphase cells and each of the seven to eight septin family members expressed by the two analyzed cell types appears equally represented. However, studies of myeloid and lymphoid cell model systems revealed cell type–specific alterations of higher-order septin arrangements in response to substrate adhesion. Live-cell observations suggested that all higher-order septin assemblies are mutually exclusive with plasma membrane regions undergoing remodeling. The combined data point to a mechanism by which densely arranged cortical microtubules, which are typical for nonadhered spherical cells, support plasma membrane–bound, disk-like septin assemblies.

scholarly journals Mechanotransduction in an extracted cell model: Fyn drives stretch- and flow-elicited PECAM-1 phosphorylation

2008 ◽  
Vol 182 (4) ◽  
pp. 753-763 ◽  
Author(s):  
Yi-Jen Chiu ◽  
Elena McBeath ◽  
Keigi Fujiwara
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Adhesion Molecule ◽  
Mammalian Cells ◽  
Kinase Inhibitors ◽  
Vascular Endothelial Cells ◽  
Cell Model ◽  
Vascular Endothelial ◽  
Small Interfering Rnas ◽  
Essential Components

Mechanosensing followed by mechanoresponses by cells is well established, but the mechanisms by which mechanical force is converted into biochemical events are poorly understood. Vascular endothelial cells (ECs) exhibit flow- and stretch-dependent responses and are widely used as a model for studying mechanotransduction in mammalian cells. Platelet EC adhesion molecule 1 (PECAM-1) is tyrosine phosphorylated when ECs are exposed to flow or when PECAM-1 is directly pulled, suggesting that it is a mechanochemical converter. We show that PECAM-1 phosphorylation occurs when detergent-extracted EC monolayers are stretched, indicating that this phosphorylation is mechanically triggered and does not require the intact plasma membrane and soluble cytoplasmic components. Using kinase inhibitors and small interfering RNAs, we identify Fyn as the PECAM-1 kinase associated with the model. We further show that stretch- and flow-induced PECAM-1 phosphorylation in intact ECs is abolished when Fyn expression is down-regulated. We suggest that PECAM-1 and Fyn are essential components of a PECAM-1–based mechanosensory complex in ECs.

Molecular mechanism of secretory vesicle docking

2010 ◽  
Vol 38 (1) ◽  
pp. 192-198 ◽  
Author(s):  
Heidi de Wit
Keyword(s):  
Plasma Membrane ◽  
Cell Model ◽  
Secretory Vesicle ◽  
Model Systems ◽  
Secretory Vesicles ◽  
Filamentous Actin ◽  
Embryonic Mouse ◽  
Vesicle Docking ◽  
Bovine Chromaffin Cell ◽  
Stable Association

Docking, the stable association of secretory vesicles with the plasma membrane, is considered to be the necessary first step before vesicles gain fusion-competence, but it is unclear how vesicles dock. In adrenal medullary chromaffin cells, access of secretory vesicles to docking sites is controlled by dense F-actin (filamentous actin) beneath the plasma membrane. Recently, we found that, in the absence of Munc18-1, the number of docked vesicles and the thickness of cortical F-actin are affected. In the present paper, I discuss the possible mechanism by which Munc18-1 modulates cortical F-actin and how it orchestrates the docking machinery via an interaction with syntaxin-1. Finally, a comparison of Munc18's role in embryonic mouse and adult bovine chromaffin cell model systems will be made to clarify observed differences in cortical F-actin as well as docking phenotypes.

scholarly journals Pexophagy: The Selective Degradation of Peroxisomes

2012 ◽  
Vol 2012 ◽  
pp. 1-18 ◽  
Author(s):  
Andreas Till ◽  
Ronak Lakhani ◽  
Sarah F. Burnett ◽  
Suresh Subramani
Keyword(s):  
Mammalian Cells ◽  
Current Knowledge ◽  
Cell Types ◽  
Model Systems ◽  
Eukaryotic Cells ◽  
Organelle Biogenesis ◽  
Selective Degradation ◽  
Future Challenges ◽  
Recent Developments ◽  
Distinct Features

Peroxisomes are single-membrane-bounded organelles present in the majority of eukaryotic cells. Despite the existence of great diversity among different species, cell types, and under different environmental conditions, peroxisomes contain enzymes involved inβ-oxidation of fatty acids and the generation, as well as detoxification, of hydrogen peroxide. The exigency of all eukaryotic cells to quickly adapt to different environmental factors requires the ability to precisely and efficiently control peroxisome number and functionality. Peroxisome homeostasis is achieved by the counterbalance between organelle biogenesis and degradation. The selective degradation of superfluous or damaged peroxisomes is facilitated by several tightly regulated pathways. The most prominent peroxisome degradation system uses components of the general autophagy core machinery and is therefore referred to as “pexophagy.” In this paper we focus on recent developments in pexophagy and provide an overview of current knowledge and future challenges in the field. We compare different modes of pexophagy and mention shared and distinct features of pexophagy in yeast model systems, mammalian cells, and other organisms.

scholarly journals Trypanosoma cruzi: mechanism of entry and intracellular fate in mammalian cells.

1976 ◽  
Vol 143 (6) ◽  
pp. 1402-1420 ◽  
Author(s):  
N Nogueira ◽  
Z Cohn
Keyword(s):  
Plasma Membrane ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Cytoplasmic Matrix ◽  
Mode Of Entry ◽  
Intracellular Fate

The mode of entry and intracellular fate of epimastigotes and trypomastigotes of Trypanosoma cruzi in cultured cells was studied. Electron microscopic observations indicated the uptake by phagocytosis of both forms into mouse peritoneal macrophages and of trypomastigotes and transition forms into other cultured cell types. In each instance the organisms were initially surrounded by a plasma membrane-derived phagosome. Trypsin and chymotrypsin treatment of the macrophages completely abolished attachment and ingestion of both forms, indicating that protease-sensitive structures on the macrophage plasma membrane mediate ingestion. The macrophage Fc or C3b receptors were not essential for uptake of T. cruzi in the conditions used. Cytochalasin B inhibited ingestion but not the attachment of both forms by macrophages. Epimastigotes were not taken up by HeLa, L cells, and calf embryo fibroblasts. In macrophages, epimastigotes were killed and digested within phagolysosomes. In contrast, trypomastigotes and transition forms escaped from the phagocytic vacuole and then multiplied in the cytoplasmic matrix. Amastigotes released from infected cells exhibited properties similar to those of trypomastigotes and were able to enter all cell types studied and multiply intracellularly.

Structural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes

1997 ◽  
Vol 110 (16) ◽  
pp. 1893-1906 ◽  
Author(s):  
R.M. Golsteyn ◽  
M.C. Beckerle ◽  
T. Koay ◽  
E. Friederich
Keyword(s):  
Plasma Membrane ◽  
Listeria Monocytogenes ◽  
Mammalian Cells ◽  
Chimeric Protein ◽  
Cell Types ◽  
Striking Similarity ◽  
Rich Domain ◽  
Protein Partners ◽  
Localization Sequence

The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement. The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells. Therefore we examined the possibility that an ActA-like protein is present in mammalian cells. By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types. We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box). Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences. A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells. Furthermore, zyxin and ActA exhibit common protein partners in vitro. On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells.

Shank2E binds NaPi cotransporter at the apical membrane of proximal tubule cells

2005 ◽  
Vol 289 (4) ◽  
pp. C1042-C1051 ◽  
Author(s):  
Ryan R. McWilliams ◽  
Sophia Y. Breusegem ◽  
Kelley F. Brodsky ◽  
Eunjoon Kim ◽  
Moshe Levi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Apical Membrane ◽  
Cell Model ◽  
Rat Kidney ◽  
Pdz Domain ◽  
Cell Types ◽  
Significant Degree ◽  
Ok Cells ◽  
Apical Domain

Proteins expressing postsynaptic density (PSD)-95/ Drosophila disk large (Dlg)/zonula occludens-1 (ZO-1) (PDZ) domains are commonly involved in moderating receptor, channel, and transporter activities at the plasma membrane in a variety of cell types. At the apical membrane of renal proximal tubules (PT), the type IIa NaPi cotransporter (NaPi-IIa) binds specific PDZ domain proteins. Shank2E is a spliceoform of a family of PDZ proteins that is concentrated at the apical domain of liver and pancreatic epithelial cell types and is expressed in kidney. In the present study, immunoblotting of enriched plasma membrane fractions and immunohistology found Shank2E concentrated at the brush border membrane of rat PT cells. Confocal localization of Flag-Shank2E and enhanced green fluorescent protein-NaPi-IIa in cotransfected OK cells showed these proteins colocalized in the apical microvilli of this PT cell model. Shank2E coimmunoprecipitated with NaPi-IIa from rat renal cortex tissue and HA-NaPi-IIa coprecipitated with Flag-Shank2E in cotransfected human embryonic kidney HEK cells. Domain analysis showed that the PDZ domain of Shank2E specifically bound NaPi-IIa and truncation of the COOH-terminal TRL motif from NaPi-IIa abolished this binding, and Far Western blotting showed that the Shank2E- NaPi-IIa interaction occurred directly between the two proteins. NaPi-IIa activity is regulated by moderating its abundance in the apical membrane. High-Pi conditions induce NaPi-IIa internalization and degradation. In both rat kidney PT cells and OK cells, shifting to high-Pi conditions induced an acute internal redistribution of Shank2E and, in OK cells, a significant degree of degradation. In sum, Shank2E is concentrated in the apical domain of renal PT cells, specifically binds NaPi-IIa via PDZ interactions, and undergoes Pi-induced internalization.

scholarly journals A Polybasic Domain in aPKC Mediates Par6-Dependent Control of Membrane Targeting and Kinase Activity

2019 ◽  
Author(s):  
Wei Dong ◽  
Juan Lu ◽  
Xuejing Zhang ◽  
Yan Wu ◽  
Kaela Lettieri ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Protein Interactions ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Allosteric Regulation ◽  
Cell Polarization ◽  
Physical Interaction ◽  
Binding Domains

SUMMARYMechanisms coupling the atypical PKC (aPKC) kinase activity to its subcellular localization are essential for cell polarization. Unlike other members of the PKC family, aPKC has no well-defined plasma membrane (PM) or calcium binding domains, leading to the assumption that its subcellular localization relies exclusively on protein-protein interactions. Here we show that in both Drosophila and mammalian cells the pseudosubstrate region (PSr) of aPKC acts as a polybasic domain capable of targeting aPKC to the PM via electrostatic binding to PM PI4P and PI(4,5)P2. However, physical interaction between aPKC and Par-6 is required for the PM-targeting of aPKC, likely by allosterically exposing the PSr to bind PM. Binding of Par-6 also inhibits aPKC kinase activity and such inhibition can be relieved through Par-6 interaction with apical polarity protein Crumbs. Our data suggest a potential mechanism in which allosteric regulation of polybasic PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of its kinase activity.eTOC SummaryDong et al. discover that the pseudo-substrate region (PSr) in aPKC is a polybasic domain capable of electrostatically targeting aPKC to plasma membrane. Allosteric regulation of PSr by Par-6 couples the control of both aPKC subcellular localization and spatial activation of kinase activity.

scholarly journals Sphingomyelinase Treatment Induces ATP-independent Endocytosis

1998 ◽  
Vol 140 (1) ◽  
pp. 39-47 ◽  
Author(s):  
Xiaohui Zha ◽  
Lynda M. Pierini ◽  
Philip L. Leopold ◽  
Paul J. Skiba ◽  
Ira Tabas ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Fluid Phase ◽  
Cell Types ◽  
Transferrin Receptors ◽  
General Requirement ◽  
Late Endosomes ◽  
Hydrolysis Of

ATP hydrolysis has been regarded as a general requirement for internalization processes in mammalian cells. We found, however, that treatment of ATP-depleted macrophages and fibroblasts with exogenous sphingomyelinase (SMase) rapidly induces formation of numerous vesicles that pinch off from the plasma membrane; the process is complete within 10 min after adding SMase. By electron microscopy, the SMase-induced vesicles are ∼400 nm in diameter and lack discernible coats. 15–30% of plasma membrane is internalized by SMase treatment, and there is no detectable enrichment of either clathrin or caveolin in these vesicles. When ATP is restored to the cells, the SMase-induced vesicles are able to deliver fluid-phase markers to late endosomes/lysosomes and return recycling receptors, such as transferrin receptors, back to the plasma membrane. We speculate that hydrolysis of sphingomyelin on the plasma membrane causes inward curvature and subsequent fusion to form sealed vesicles. Many cell types express a SMase that can be secreted or delivered to endosomes and lysosomes. The hydrolysis of sphingomyelin by these enzymes is activated by several signaling pathways, and this may lead to formation of vesicles by the process described here.

scholarly journals Modeling cell-substrate de-adhesion dynamics under fluid shear

2017 ◽  
Author(s):  
Renu Maan ◽  
Garima Rani ◽  
Gautam I. Menon ◽  
Pramod A. Pullarkat
Keyword(s):  
Shear Stress ◽  
Cell Adhesion ◽  
Cancer Metastasis ◽  
Cell Types ◽  
Model Systems ◽  
Cell Detachment ◽  
Fluid Shear ◽  
Substrate Adhesion ◽  
Cell Substrate ◽  
Cell Substrate Adhesion

AbstractChanges in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as for the decay in the fraction of detached cells at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of ~ 500 pN associated to a single focal contact, and characteristic time-scales of 190 ≤ τ ≤ 350s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

scholarly journals Global Regulation of the Interphase Microtubule System by Abundantly Expressed Op18/Stathmin

2008 ◽  
Vol 19 (7) ◽  
pp. 2897-2906 ◽  
Author(s):  
Mikael E. Sellin ◽  
Per Holmfeldt ◽  
Sonja Stenmark ◽  
Martin Gullberg
Keyword(s):  
Posttranscriptional Regulation ◽  
Cell Model ◽  
K562 Cells ◽  
Cell Types ◽  
Jurkat Cells ◽  
Model Systems ◽  
Regulatory Function ◽  
Mrna Levels ◽  
Global Regulation ◽  
Tubulin Mrna

Op18/stathmin (Op18), a conserved microtubule-depolymerizing and tubulin heterodimer-binding protein, is a major interphase regulator of tubulin monomer–polymer partitioning in diverse cell types in which Op18 is abundant. Here, we addressed the question of whether the microtubule regulatory function of Op18 includes regulation of tubulin heterodimer synthesis. We used two human cell model systems, K562 and Jurkat, combined with strategies for regulatable overexpression or depletion of Op18. Although Op18 depletion caused extensive overpolymerization and increased microtubule content in both cell types, we did not detect any alteration in polymer stability. Interestingly, however, we found that Op18 mediates positive regulation of tubulin heterodimer content in Jurkat cells, which was not observed in K562 cells. By analysis of cells treated with microtubule-poisoning drugs, we found that Jurkat cells regulate tubulin mRNA levels by a posttranscriptional mechanism similarly to normal primary cells, whereas this mechanism is nonfunctional in K562 cells. We present evidence that Op18 mediates posttranscriptional regulation of tubulin mRNA in Jurkat cells through the same basic autoregulatory mechanism as microtubule-poisoning drugs. This, combined with potent regulation of tubulin monomer–polymer partitioning, enables Op18 to exert global regulation of the microtubule system.

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