scholarly journals C-terminal phosphorylation modulates ERM-1 localization and dynamics to control cortical actin organization and support lumen formation during Caenorhabditiselegans development

Development ◽  
2020 ◽  
Vol 147 (14) ◽  
pp. dev188011 ◽  
Author(s):  
João J. Ramalho ◽  
Jorian J. Sepers ◽  
Ophélie Nicolle ◽  
Ruben Schmidt ◽  
Janine Cravo ◽  
...  
Keyword(s):  
Fine Tuning ◽  
Tissue Formation ◽  
Cortical Actin ◽  
Dynamic Regulation ◽  
Tissue Specific ◽  
Lumen Formation ◽  
Erm Proteins ◽  
Actin Organization ◽  
Apical Localization

ABSTRACTERM proteins are conserved regulators of cortical membrane specialization that function as membrane-actin linkers and molecular hubs. The activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2 binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single Caenorhabditiselegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo. Using CRISPR/Cas9-generated erm-1 mutant alleles, we demonstrate that a PIP2-binding site is crucially required for ERM-1 function. By contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.

scholarly journals In vivo control of the ezrin/radixin/moesin protein ERM-1 in C. elegans

2020 ◽  
Author(s):  
João J. Ramalho ◽  
Ophélie Nicolle ◽  
Grégoire Michaux ◽  
Mike Boxem
Keyword(s):  
Fine Tuning ◽  
Tissue Formation ◽  
Cortical Actin ◽  
Dynamic Regulation ◽  
Tissue Specific ◽  
Erm Proteins ◽  
Actin Organization ◽  
C Elegans ◽  
Apical Localization

AbstractERM proteins are conserved regulators of cortical membrane specialization, that function as membrane–actin linkers and molecular hubs. Activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2-binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single C. elegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo. Using CRISPR/Cas9-generated erm-1 mutant alleles we demonstrate that PIP2-binding is critically required for ERM-1 function. In contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and drive lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.

scholarly journals End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis in Saccharomyces cerevisiae

1997 ◽  
Vol 8 (11) ◽  
pp. 2291-2306 ◽  
Author(s):  
A. Wesp ◽  
L. Hicke ◽  
J. Palecek ◽  
R. Lombardi ◽  
T. Aust ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
High Temperature ◽  
Mutational Analysis ◽  
Coiled Coil ◽  
Temperature Sensitive ◽  
Cortical Actin ◽  
Actin Organization ◽  
Coiled Coil Domain ◽  
Associated Proteins

end4–1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) ofABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

scholarly journals Syndapin Isoforms Participate in Receptor-Mediated Endocytosis and Actin Organization

2000 ◽  
Vol 148 (5) ◽  
pp. 1047-1062 ◽  
Author(s):  
Britta Qualmann ◽  
Regis B. Kelly
Keyword(s):  
Splice Variants ◽  
Neuroendocrine Cells ◽  
Actin Dynamics ◽  
Cell Periphery ◽  
Cortical Actin ◽  
Actin Organization ◽  
Vesicle Recycling ◽  
Src Homology ◽  
Dynamin I

Syndapin I (SdpI) interacts with proteins involved in endocytosis and actin dynamics and was therefore proposed to be a molecular link between the machineries for synaptic vesicle recycling and cytoskeletal organization. We here report the identification and characterization of SdpII, a ubiquitously expressed isoform of the brain-specific SdpI. Certain splice variants of rat SdpII in other species were named FAP52 and PACSIN 2. SdpII binds dynamin I, synaptojanin, synapsin I, and the neural Wiskott-Aldrich syndrome protein (N-WASP), a stimulator of Arp2/3 induced actin filament nucleation. In neuroendocrine cells, SdpII colocalizes with dynamin, consistent with a role for syndapin in dynamin-mediated endocytic processes. The src homology 3 (SH3) domain of SdpI and -II inhibited receptor-mediated internalization of transferrin, demonstrating syndapin involvement in endocytosis in vivo. Overexpression of full-length syndapins, but not the NH2-terminal part or the SH3 domains alone, had a strong effect on cortical actin organization and induced filopodia. This syndapin overexpression phenotype appears to be mediated by the Arp2/3 complex at the cell periphery because it was completely suppressed by coexpression of a cytosolic COOH-terminal fragment of N-WASP. Consistent with a role in actin dynamics, syndapins localized to sites of high actin turnover, such as filopodia tips and lamellipodia. Our results strongly suggest that syndapins link endocytosis and actin dynamics.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

ERM proteins and NF2 tumor suppressor: the Yin and Yang of cortical actin organization and cell growth signaling

2002 ◽  
Vol 14 (1) ◽  
pp. 104-109 ◽  
Author(s):  
Alexis Gautreau ◽  
Daniel Louvard ◽  
Monique Arpin
Keyword(s):  
Cell Growth ◽  
Tumor Suppressor ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Actin Organization ◽  
Yin And Yang

scholarly journals Swf1p, a Member of the DHHC-CRD Family of Palmitoyltransferases, Regulates the Actin Cytoskeleton and Polarized Secretion Independently of Its DHHC Motif

2008 ◽  
Vol 19 (10) ◽  
pp. 4454-4468 ◽  
Author(s):  
Shubha A. Dighe ◽  
Keith G. Kozminski
Keyword(s):  
Actin Cytoskeleton ◽  
Rho Gtpase ◽  
Wild Type ◽  
Cortical Actin ◽  
Actin Organization ◽  
Bud Emergence ◽  
Polarized Secretion ◽  
Polarized Cell Growth ◽  
Actin Cables

Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton.

scholarly journals In vivo roles for Arp2/3 in cortical actin organization during C. elegans gastrulation

2009 ◽  
Vol 122 (21) ◽  
pp. 3983-3993 ◽  
Author(s):  
M. Roh-Johnson ◽  
B. Goldstein
Keyword(s):  
Cortical Actin ◽  
Actin Organization ◽  
C Elegans

Cortical actin organization is required for internalization of bombesin/GRP and EGF receptors, and ERK activation in Swiss 3T3 cells

2001 ◽  
Vol 120 (5) ◽  
pp. A310-A311
Author(s):  
J LUNN ◽  
H WONG ◽  
E ROZENGURT ◽  
J WALSH
Keyword(s):  
Egf Receptors ◽  
3T3 Cells ◽  
Cortical Actin ◽  
Erk Activation ◽  
Actin Organization ◽  
Swiss 3T3

scholarly journals 99mTc Labelling Strategies for the Development of Potential Nitroimidazolic Hypoxia Imaging Agents

Inorganics ◽  
2019 ◽  
Vol 7 (11) ◽  
pp. 128 ◽  
Author(s):  
Giglio ◽  
Rey
Keyword(s):  
Rational Design ◽  
Biological Properties ◽  
Fine Tuning ◽  
Oxidation States ◽  
Hypoxia Imaging ◽  
Biological Target ◽  
99Mtc Radiopharmaceuticals ◽  
99Mtc Labelling

Technetium-99m has a rich coordination chemistry that offers many possibilities in terms of oxidation states and donor atom sets. Modifications in the structure of the technetium complexes could be very useful for fine tuning the physicochemical and biological properties of potential 99mTc radiopharmaceuticals. However, systematic study of the influence of the labelling strategy on the “in vitro” and “in vivo” behaviour is necessary for a rational design of radiopharmaceuticals. Herein we present a review of the influence of the Tc complexes’ molecular structure on the biodistribution and the interaction with the biological target of potential nitroimidazolic hypoxia imaging radiopharmaceuticals presented in the literature from 2010 to the present. Comparison with the gold standard [18F]Fluoromisonidazole (FMISO) is also presented.

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