scholarly journals Agonist-induced PIP2 Hydrolysis Inhibits Cortical Actin Dynamics: Regulation at a Global but not at a Micrometer Scale

2002 ◽  
Vol 13 (9) ◽  
pp. 3257-3267 ◽  
Author(s):  
Jacco van Rheenen ◽  
Kees Jalink
Keyword(s):  
Actin Dynamics ◽  
Membrane Microdomains ◽  
Imaging Method ◽  
Axial Resolution ◽  
Pleckstrin Homology ◽  
Cortical Actin ◽  
Spatial Differences ◽  
Phosphatidylinositol 4 ◽  
Micrometer Scale ◽  
Homology Domains

Phosphatidylinositol 4, 5-bisphosphate (PIP2) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP2 sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP2-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at ∼15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP2 breakdown, and it resumes as soon as PIP2levels are back to normal. Thus, our data support a role for PIP2 in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP2regulation of the cytoskeleton exist at a micrometer scale.

scholarly journals PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER–PM contact sites

2018 ◽  
Vol 217 (5) ◽  
pp. 1797-1813 ◽  
Author(s):  
Mira Sohn ◽  
Marek Korzeniowski ◽  
James P. Zewe ◽  
Rachel C. Wills ◽  
Gerald R.V. Hammond ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Dynamic Control ◽  
Pleckstrin Homology ◽  
Contact Sites ◽  
Transfer Activity ◽  
Powerful Means ◽  
Phosphatidylinositol 4 ◽  
Homology Domains

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)–resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2. Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2. Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase–mediated conversion to PI(4,5)P2. Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

scholarly journals Coherence tomography with broad bandwidth extreme ultraviolet and soft X-ray radiation

2021 ◽  
Vol 127 (4) ◽  
Author(s):  
S. Skruszewicz ◽  
S. Fuchs ◽  
J. J. Abel ◽  
J. Nathanael ◽  
J. Reinhard ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Near Infrared ◽  
Extreme Ultraviolet ◽  
Infrared Light ◽  
Imaging Method ◽  
Optical Coherence ◽  
Axial Resolution ◽  
Cross Sectional ◽  
X Ray ◽  
Broad Bandwidth

AbstractWe present an overview of recent results on optical coherence tomography with the use of extreme ultraviolet and soft X-ray radiation (XCT). XCT is a cross-sectional imaging method that has emerged as a derivative of optical coherence tomography (OCT). In contrast to OCT, which typically uses near-infrared light, XCT utilizes broad bandwidth extreme ultraviolet (XUV) and soft X-ray (SXR) radiation (Fuchs et al in Sci Rep 6:20658, 2016). As in OCT, XCT’s axial resolution only scales with the coherence length of the light source. Thus, an axial resolution down to the nanometer range can be achieved. This is an improvement of up to three orders of magnitude in comparison to OCT. XCT measures the reflected spectrum in a common-path interferometric setup to retrieve the axial structure of nanometer-sized samples. The technique has been demonstrated with broad bandwidth XUV/SXR radiation from synchrotron facilities and recently with compact laboratory-based laser-driven sources. Axial resolutions down to 2.2 nm have been achieved experimentally. XCT has potential applications in three-dimensional imaging of silicon-based semiconductors, lithography masks, and layered structures like XUV mirrors and solar cells.

Pleckstrin homology domains: not just for phosphoinositides

2004 ◽  
Vol 32 (5) ◽  
pp. 707-711 ◽  
Author(s):  
M.A. Lemmon
Keyword(s):  
Subcellular Localization ◽  
Human Genome ◽  
Small Gtpases ◽  
Membrane Targeting ◽  
Ph Domain ◽  
Cellular Membranes ◽  
Pleckstrin Homology ◽  
Ph Domains ◽  
Common Domain ◽  
Homology Domains

PH domains (pleckstrin homology domains) are the 11th most common domain in the human genome and are best known for their ability to target cellular membranes by binding specifically to phosphoinositides. Recent studies in yeast have shown that, in fact, this is a property of only a small fraction of the known PH domains. Most PH domains are not capable of independent membrane targeting, and those capable of doing so (approx. 33%) appear, most often, to require both phosphoinositide and non-phosphoinositide determinants for their subcellular localization. Several recent studies have suggested that small GTPases such as ARF family proteins play a role in defining PH domain localization. Some others have described a signalling role for PH domains in regulating small GTPases, although phosphoinositides may also play a role. These findings herald a change in our perspective of PH domain function, which will be significantly more diverse than previously supposed.

scholarly journals Control of cortical cytoskeleton-membrane interaction by RhoA regulates peripheral nerve myelination

2021 ◽  
Author(s):  
Ana I. Seixas ◽  
Miguel R. G. Morais ◽  
Cord Brakebusch ◽  
Jo&atildeo B. Relvas
Keyword(s):  
Schwann Cell ◽  
Schwann Cells ◽  
Rho Gtpases ◽  
Intracellular Signaling ◽  
Cell Interaction ◽  
Actin Dynamics ◽  
Specific Gene ◽  
Cortical Actin ◽  
Membrane Attachment ◽  
Cytoskeleton Dynamics

Bidirectional transmission of mechanical and biochemical signals is integral to cell-environment communication and underlies the function of Schwann cells, the myelinating glia of the peripheral nervous system. As major integrators of "outside-in" signaling, Rho GTPases link actin cytoskeleton dynamics with cellular architecture to regulate adhesion and cell deformation. Using Schwann cell-specific gene inactivation, we discovered that RhoA promotes the initiation of myelination, axonal wrapping and axial spreading of Schwann cells, and is later required to restrict myelin growth in peripheral nerves. These effects are mediated by modulation of actomyosin contractility, actin dynamics and cortical actin-membrane attachment, which collectively couple tensional forces to intracellular signaling that regulate axon-Schwann cell interaction and myelin synthesis. This work establishes RhoA as an intrinsic regulator of a biomechanical response that controls the switch of Schwann cells towards the myelinating and the homeostatic states.

scholarly journals Arp2/3 mediates early endosome dynamics necessary for the maintenance of PAR asymmetry in Caenorhabditis elegans

2012 ◽  
Vol 23 (10) ◽  
pp. 1917-1927 ◽  
Author(s):  
Jessica M. Shivas ◽  
Ahna R. Skop
Keyword(s):  
Caenorhabditis Elegans ◽  
Maintenance Phase ◽  
Early Endosome ◽  
Actin Dynamics ◽  
Cellular Polarity ◽  
Cortical Actin ◽  
Endocytic Recycling ◽  
C Elegans ◽  
Early Endosomes ◽  
Stable Maintenance

The widely conserved Arp2/3 complex regulates branched actin dynamics that are necessary for a variety of cellular processes. In Caenorhabditis elegans, the actin cytoskeleton has been extensively characterized in its role in establishing PAR asymmetry; however, the contributions of actin to the maintenance of polarity before the onset of mitosis are less clear. Endocytic recycling has emerged as a key mechanism in the dynamic stabilization of cellular polarity, and the large GTPase dynamin participates in the stabilization of cortical polarity during maintenance phase via endocytosis in C. elegans. Here we show that disruption of Arp2/3 function affects the formation and localization of short cortical actin filaments and foci, endocytic regulators, and polarity proteins during maintenance phase. We detect actin associated with events similar to early endosomal fission, movement of endosomes into the cytoplasm, and endosomal movement from the cytoplasm to the plasma membrane, suggesting the involvement of actin in regulating processes at the early endosome. We also observe aberrant accumulations of PAR-6 cytoplasmic puncta near the centrosome along with early endosomes. We propose a model in which Arp2/3 affects the efficiency of rapid endocytic recycling of polarity cues that ultimately contributes to their stable maintenance.

Sla1p couples the yeast endocytic machinery to proteins regulating actin dynamics

2002 ◽  
Vol 115 (8) ◽  
pp. 1703-1715 ◽  
Author(s):  
Derek T. Warren ◽  
Paul D. Andrews ◽  
Campbell W. Gourlay ◽  
Kathryn R. Ayscough
Keyword(s):  
Immunofluorescence Microscopy ◽  
Fluid Phase ◽  
Actin Dynamics ◽  
Cell Cortex ◽  
Cortical Actin ◽  
Binding Studies ◽  
Endocytic Machinery ◽  
Single Complex ◽  
Actin Patch ◽  
First Time

Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p(YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.

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