Microinjection of Fluorescently Labeled 130K Protein into Living Fibroblasts: Localization at the Ends of Actin Microfilament Bundles and in Close Relation to Fibronectin

1980 ◽  
Vol 38 ◽  
pp. 708-711
Author(s):  
J. R. Feramisco ◽  
K. Burridge
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Actin Filaments ◽  
Cell Biology ◽  
Close Relation ◽  
Cell Types ◽  
Living Cells ◽  
Structural Proteins ◽  
Major Goal ◽  
Microfilament Bundles

Understanding the interactions between cell surface components and the underlying cytoskeleton continues to be a major goal in cell biology. In particular, little is known about the molecules involved in linking actin filaments to the plasma membrane in cells such as fibroblasts. A protein with a molecular weight of 130,000 (the 130K protein) was recently purified from smooth muscle by Geiger and localized to the ends of microfilament bundles in a variety of cell types. Independently we had purified this protein and were studying its possible interactions with other structural proteins when we learned of its interesting location, one that would be consistent with some role in the attachment of actin filaments to the plasma membrane. We were prompted to investigate this interesting protein further by microinjection of the f1uorescent1y labeled protein into living cells, a technique which had recently been successfully applied to the study of α-actinin.

Proteins involved in the attachment of actin to the plasma membrane

1982 ◽  
Vol 299 (1095) ◽  
pp. 291-299 ◽  
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Cell Types ◽  
Brain Protein ◽  
Cultured Fibroblasts ◽  
Microfilament Bundles ◽  
Different Cell Types ◽  
Adhesion Plaque ◽  
The Brain

Proteins that may be involved in two types of actin-membrane association are discussed. The first set includes α-actinin, vinculin, fimbrin and a new cytoskeletal protein that are all concentrated in adhesion plaques, those regions of cultured fibroblasts where bundles of actin microfilaments terminate and where the plasma membrane comes close to the underlying substrate. The properties of non-muscle α-actinin suggest that it functions to cross-link actin filaments and thereby stabilize microfilament bundles rather than functioning in their attachment to the membrane. Fimbrin also appears to be involved in bundling of filaments rather than in attachment. In contrast, vinculin binds to the ends of actin filaments in vitro and is probably the best candidate for a role in the attachment of actin to membranes at the adhesion plaque. The discovery of a new protein, 215k, of unknown function, in the adhesion plaque suggests that many more proteins remain to be identified in this region. Attachment of actin filaments to other regions of the plasma membrane is also considered and a protein is described that seems to be a spectrin homologue in brain and other tissues. The brain protein resembles erythrocyte spectrin in its physical properties, in binding actin, in being associated with cell membranes and in crossreacting immunologically. We suggest that the brain protein and erythrocyte spectrin both belong to a family of related proteins (the spectrins) which function in the attachment of actin to membranes in many different cell types.

scholarly journals Actin-membrane interaction in fibroblasts: what proteins are involved in this association?

1984 ◽  
Vol 99 (1) ◽  
pp. 95s-103s ◽  
Author(s):  
P Mangeat ◽  
K Burridge
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Stress Fiber ◽  
Membrane Interaction ◽  
Microfilament Bundles ◽  
The Family ◽  
Form Part ◽  
Membrane Attachment ◽  
A Chain ◽  
And Function

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.

scholarly journals The distinguishing characteristics of the plasma membrane are its exposed proteins

1975 ◽  
Vol 147 (2) ◽  
pp. 373-376 ◽  
Author(s):  
R E Gates ◽  
D R Phillips ◽  
M Morrison
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Human Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Types ◽  
Distinguishing Characteristic ◽  
Probe System ◽  
Normal Human

The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.

scholarly journals Real-time measurement of E2:ERα transcriptional activity in living cells

2019 ◽  
Author(s):  
Manuela Cipolletti ◽  
Stefano Leone ◽  
Stefania Bartoloni ◽  
Claudia Busonero ◽  
Filippo Acconcia
Keyword(s):  
Plasma Membrane ◽  
Real Time ◽  
Cell Biology ◽  
Transcriptional Activity ◽  
Receptor Activation ◽  
Living Cells ◽  
Membrane Localization ◽  
Dependent Manner ◽  
Plasma Membrane Localization ◽  
The Impact

AbstractKinetic analyses of diverse physiological processes have the potential to unveil new aspects of the molecular regulation of cell biology at temporal levels. 17β-estradiol (E2) regulates diverse physiological effects by binding to the estrogen receptor α (ERα), which primarily works as a transcription factor. Although many molecular details of the modulation of ERα transcriptional activity have been discovered including the impact of receptor plasma membrane localization and its relative E2-evoked signalling, the knowledge of real-time ERα transcriptional dynamics in living cells is lacking. Here, we report the generation of MCF-7 and HeLa cells stably expressing a modified luciferase under the control of an E2-sensitive promoter, which activity can be continuously monitored in living cells and show that E2 induces a linear increase in ERα transcriptional activity. Ligand-independent (e.g., epidermal growth factor) receptor activation was also detected in a time-dependent manner. Kinetic profiles of ERα transcriptional activity measured in the presence of both receptor antagonists and inhibitors of ERα plasma membrane localization reveals a biphasic dynamic of receptor behaviour underlying novel aspects of receptor-regulated transcriptional effects. Finally, analysis of the rate of the dose-dependent E2 induction of ERα transcriptional activity demonstrates that low doses of E2 induce an effect identical to that determined by high concentrations of E2 as a function of the duration of hormone administration. Overall, we present the characterization of sensitive stable cell lines where to study the kinetic of E2 transcriptional signaling and to identify new aspects of ERα function in different physiological or pathophysiological conditions.

scholarly journals An agent-based model of molecular aggregation at the cell membrane

2019 ◽  
Author(s):  
Juliette Griffié ◽  
Ruby Peters ◽  
Dylan M. Owen
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Super Resolution ◽  
Cell Types ◽  
Signalling Pathways ◽  
Molecular Aggregation ◽  
Agent Based Model ◽  
Agent Based ◽  
Molecular Clustering ◽  
In Cells

AbstractMolecular clustering at the plasma membrane has long been identified as a key process and is associated with regulating signalling pathways across cell types. Recent advances in microscopy, in particular the rise of super-resolution, have allowed the experimental observation of nanoscale molecular clusters in the plasma membrane. However, modelling approaches capable of recapitulating these observations are in their infancy, partly because of the extremely complex array of biophysical factors which influence molecular distributions and dynamics in the plasma membrane. We propose here a highly abstracted approach: an agent-based model dedicated to the study of molecular aggregation at the plasma membrane. We show that when molecules are modelled as though they can act (diffuse) in a manner which is influenced by their molecular neighbourhood, many of the distributions observed in cells can be recapitulated, even though such sensing and response is not possible for real membrane molecules. As such, agent-based offers a unique platform which may lead to a new understanding of how molecular clustering in extremely complex molecular environments can be abstracted, simulated and interpreted using simple rules.Author summaryMolecular aggregation in cell membranes is a key component of cellular machinery, involved across cell types in inter-cellular communication and signalling pathway initiation. As such, understanding the underlying mechanisms and molecule cluster characteristics at a more theoretical level is a pre-requisite. Complete descriptive molecular models have proven impossible to realise due to the overall complexity of the processes involved, highlighting the need for novel approaches. While conceptual models have been shown to be powerful tools and are routinely used in other fields with high level of complexity such as social sciences or economics, they are overall lacking from the literature when it comes to cell studies. We suggest in this work that the same principle applies to cell biology and in particular, the study of molecular clustering. We propose here a general model, independent of cell types or signalling pathways: an agent-based model dedicated to molecular clustering in the plasma membrane. We show we are able to recapitulate molecular aggregation similar to observations in cells while new properties are highlighted by our model, for instance, clustering is a digitised process.

Reversible - through calmodulin - electrostatic interactions between basic residues on proteins and acidic lipids in the plasma membrane.

2005 ◽  
Vol 72 ◽  
pp. 189-198 ◽  
Author(s):  
Stuart McLaughlin ◽  
Gyöngyi Hangyás-Mihályné ◽  
Irina Zaitseva ◽  
Urszula Golebiewska
Keyword(s):  
Plasma Membrane ◽  
Cell Biology ◽  
Electrostatic Interactions ◽  
Theoretical Calculations ◽  
Cell Types ◽  
Kinase Substrate ◽  
Poisson Boltzmann ◽  
Pkc Protein Kinase C ◽  
Poisson Boltzmann Equation ◽  
Acidic Lipids

The inner leaflet of a typical mammalian plasma membrane contains 20-30% univalent PS (phosphatidylserine) and 1% multivalent PtdIns(4,5)P2. Numerous proteins have clusters of basic (or basic/hydrophobic) residues that bind to these acidic lipids. The intracellular effector CaM (calmodulin) can reverse this binding on a wide variety of proteins, including MARCKS (myristoylated alanine-rich C kinase substrate), GAP43 (growth-associated protein 43, also known as neuromodulin), gravin, GRK5 (G-protein-coupled receptor kinase 5), the NMDA (N-methyl-d-aspartate) receptor and the ErbB family. We used the first principles of physics, incorporating atomic models and the Poisson-Boltzmann equation, to describe how the basic effector domain of MARCKS binds electrostatically to acidic lipids on the plasma membrane. The theoretical calculations show the basic cluster produces a local positive electrostatic potential that should laterally sequester PtdInsP2, even when univalent acidic lipids are present at a physiologically relevant 100-fold excess; four independent experimental measurements confirm this prediction. Ca2+/CaM binds with high affinity (Kd approximately 10nM) to this domain and releases the PtdIns(4,5)P2. MARCKS, a major PKC (protein kinase C) substrate, is present at concentrations comparable with those of PtdIns(4,5)P2 (approx. 10 μM) in many cell types. Thus MARCKS can act as a reversible PtdIns(4,5)P2 buffer, binding PtdIns(4,5)P2 in a quiescent cell, and releasing it locally when the intracellular Ca2+ concentration increases. This reversible sequestration is important because PtdIns(4,5)P2 plays many roles in cell biology. Less is known about the role of CaM-mediated reversible membrane binding of basic/hydrophobic clusters for the other proteins.

Villin-induced growth of microvilli is reversibly inhibited by cytochalasin D

1993 ◽  
Vol 105 (3) ◽  
pp. 765-775 ◽  
Author(s):  
E. Friederich ◽  
T.E. Kreis ◽  
D. Louvard
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Site ◽  
Actin Filaments ◽  
Living Cells ◽  
Cytochalasin D ◽  
Actin Binding ◽  
Fast Growing ◽  
Actin Binding Site

Villin is an actin-binding protein that is associated with the cytoskeleton of brush border microvilli. In vitro, villin nucleates, caps or severs actin filaments in a Ca(2+)-dependent manner. In the absence of Ca2+, villin organizes microfilaments into bundles. Transfection of a villin-specific cDNA into cultured cells that do not produce this protein results in the growth of long surface microvilli and the reorganization of the underlying actin cytoskeleton. Here we studied the effects of low concentrations of cytochalasin D on the induction of these plasma membrane-actin cytoskeleton specializations. Transfected cells were treated with concentrations of cytochalasin D that prevent the association of actin monomers with the fast-growing end of microfilaments in vitro. In villin-positive cells, cytochalasin D inhibited the growth of microvilli and promoted the formation of rodlet-like actin structures, which were randomly distributed throughout the cytoplasm. The formation of these structures was dependent on large amounts of villin and on the integrity of an actin-binding site located at the carboxy terminus of villin, which is required for microfilament bundling in vitro and for the growth of microvilli in vivo. The effect of cytochalasin D was reversible. The observation of living cells by video-imaging revealed that when cytochalasin D was removed, rapid disassembly of actin rodlets occurred after a lag phase. The present data stress the important role of the plasma membrane in the organization of the actin cytoskeleton and suggest that the extension of the microvillar plasma membrane is dependent on the elongation of microfilaments at their fast-growing end. Inhibition of microfilament elongation near the plasma membrane by cytochalasin D may result in the ‘random’ nucleation of actin filaments throughout the cytoplasm. On the basis of the present data, we propose that villin is involved in the assembly of the microvillar actin bundle by a mechanism that does not prevent monomer association with the preferred end of microfilaments. For instance, villin may stabilize actin filaments by lateral interactions. The functional importance of the carboxy-terminal F-actin binding site in such a mechanism is stressed by the fact that it is required for the formation of F-actin rodlets in cytochalasin D-treated cells. Finally, our data further emphasize the observations that the effects of cytochalasin D in living cells can be modulated by actin-binding proteins.

scholarly journals T-cells play the classics with a different spin

2014 ◽  
Vol 25 (11) ◽  
pp. 1699-1703 ◽  
Author(s):  
Michael L. Dustin
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
Immune Cells ◽  
Cell Biology ◽  
Immunological Synapse ◽  
Cell Communication ◽  
Intraflagellar Transport ◽  
Cell Types ◽  
Immune Synapse ◽  
And Function

The immune system uses much of the classic machinery of cell biology, but in ways that put a different spin on organization and function. Striking recent examples include the demonstration of intraflagellar transport protein and hedgehog contributions to the immune synapse, even though immune cells lack a primary cilium that would be the typical setting for this machinery. In a second example, lymphocytes have their own subfamily of integrins, the β2 subfamily, and only integrins in this family form a stable adhesion ring using freely mobile ligands, a key feature of the immunological synapse. Finally, we showed recently that T-cells use endosomal sorting complexes required for transport (ESCRTs) at the plasma membrane to generate T-cell antigen receptor–enriched microvesicles. It is unusual for the ESCRT pathway to operate at the plasma membrane, but this may allow a novel form of cell–cell communication by providing a multivalent ligand for major histocompatibility complex–peptide complexes and perhaps other receptors on the partnering B-cell. Immune cells are thus an exciting system for novel cell biology even with classical pathways that have been studied extensively in other cell types.

scholarly journals Distribution of flourescently labeled α-actinin in living and fixed fibroblasts

1980 ◽  
Vol 86 (2) ◽  
pp. 608-615 ◽  
Author(s):  
Feramisco JR ◽  
SH Blose
Keyword(s):  
Living Cell ◽  
Cultured Cells ◽  
Living Cells ◽  
Stress Fibers ◽  
Structural Proteins ◽  
Periodic Pattern ◽  
Microfilament Bundles ◽  
First Time ◽  
Fluorescent Derivative ◽  
Injection Procedure

The distribution of flourescently labeled α-actinin after microinjection into fibroblasts has been determined in both living and fixed cells. We have found that the distribution of the injected tetramethylrhodamine isthiocyanate-labeled protein (TMRITC-α-actinin) in living cells, which is in ruffling membranes, actin microfilament bundles, and polygonal microfilament networks (Feramisco, 1979, Proc. Natl. Acad. Sci. U. S. A. 76:3967-3971), was virtually unaffected by the fixation (3.5 percent formaldehyde) and extraction (absolute acetone) used for the preparation of the cells for immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. Also, these patterns were found to coincide with the α-actinin revealed by immunoflourescence. These findings offer, for the first time, evidence indicating the validity of the immunoflourescence technique in the localization of α-actinin in cultured cells. With the combination of the injection procedure and the immunoflourescence localization of endogenous structural proteins, it was determined that nearly all of the actin stress fibers were decorated in a periodic manner with the injected α-actinin. Endogenous tropomyosin in the injected cells was found to be distributed with a periodic pattern along the stress fibers that was antiperiodic to the pattern observed for the microinjected α-actinin. The tropomyosin antibody stained the polygonal microfilament networks and was excluded from the foci, whereas the microinjected α-actinin was incorporated into the foci of the networks. Thus, the microinjected fluorescent derivative of α-actinin appears to be incorporated into the functional pools of α-actinin within the living cell and to be utilized by the cell with fidelity.

Protein trafficking and polarity in kidney epithelium: from cell biology to physiology

1996 ◽  
Vol 76 (1) ◽  
pp. 245-297 ◽  
Author(s):  
D. Brown ◽  
J. L. Stow
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Trafficking ◽  
Cell Biology ◽  
Cell Types ◽  
Membrane Domains ◽  
Disease States ◽  
Target Membrane ◽  
Plasma Membrane Domains ◽  
Membrane Components

The transepithelial movement of fluids, electrolytes, and larger molecules is achieved by the activity of a host of specialized transporting proteins, including enzymes, receptors, and channels, that are located on either the apical, basal, or lateral plasma membrane domains of epithelial cells. In the kidney as well as in all other organs, this remarkable polarity of epithelial cells depends on the selective insertion of newly synthesized and recycling proteins and lipids into distinct plasma membrane domains and on the maintenance and modulation of these specialized domains once they are established during epithelial development. This review addresses the mechanisms by which epithelial cells control the movement of membrane components within the cell to ensure that they are delivered to the correct target membrane. Among the topics discussed are targeting signals within membrane proteins, the role of the cytoskeleton and the tight junctional barrier in cell polarity, and the requirement for accessory proteins in the targeting process, including GTP-binding proteins, and proteins that are involved in vesicle docking and fusion events. The final part of the review is devoted uniquely to the polarized targeting of functionally defined proteins in various kidney cell types. In concluding, examples of how a breakdown in these trafficking pathways may be related to some disease states are presented.

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