Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations

2001 ◽  
Vol 79 (6) ◽  
pp. 719-728 ◽  
Author(s):  
Ralf Schindler ◽  
Elke Weichselsdorfer ◽  
Oliver Wagner ◽  
Jürgen Bereiter-Hahn
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filaments ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Actin Binding ◽  
Permeabilized Cells ◽  
Specific Regulation ◽  
Actin Fibrils ◽  
Fructose 1,6 Bisphosphate

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20–2000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20–60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin–aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.

scholarly journals Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases

2001 ◽  
Vol 355 (3) ◽  
pp. 805-817 ◽  
Author(s):  
Cindy J. O'MALLEY ◽  
Brad K. McCOLL ◽  
Anne M. KONG ◽  
Sarah L. ELLIS ◽  
A. Primrose W. WIJAYARATNAM ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Inducible Promoter ◽  
Actin Binding ◽  
Inositol Polyphosphate ◽  
Null Mutant ◽  
Phosphatase Domain ◽  
Signalling Molecules ◽  
Yeast Strains ◽  
Bud Site

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P2, forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P2 in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P2 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P2 levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P2 levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for ∆inp51∆inp53, ∆inp51∆inp52 and ∆inp52∆inp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P2 accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P2 5-phosphatases.

scholarly journals Direct Visualization of Actin Filaments and Actin-Binding Proteins in Neuronal Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Minkyo Jung ◽  
Doory Kim ◽  
Ji Young Mun
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Binding Proteins ◽  
Light And Electron Microscopy ◽  
Super Resolution ◽  
Actin Binding ◽  
Direct Visualization ◽  
Actin Binding Proteins ◽  
Synapse Plasticity

Actin networks and actin-binding proteins (ABPs) are most abundant in the cytoskeleton of neurons. The function of ABPs in neurons is nucleation of actin polymerization, polymerization or depolymerization regulation, bundling of actin through crosslinking or stabilization, cargo movement along actin filaments, and anchoring of actin to other cellular components. In axons, ABP–actin interaction forms a dynamic, deep actin network, which regulates axon extension, guidance, axon branches, and synaptic structures. In dendrites, actin and ABPs are related to filopodia attenuation, spine formation, and synapse plasticity. ABP phosphorylation or mutation changes ABP–actin binding, which regulates axon or dendritic plasticity. In addition, hyperactive ABPs might also be expressed as aggregates of abnormal proteins in neurodegeneration. Those changes cause many neurological disorders. Here, we will review direct visualization of ABP and actin using various electron microscopy (EM) techniques, super resolution microscopy (SRM), and correlative light and electron microscopy (CLEM) with discussion of important ABPs in neuron.

scholarly journals The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

2009 ◽  
Vol 20 (6) ◽  
pp. 1618-1628 ◽  
Author(s):  
Alastair S. Robertson ◽  
Ellen G. Allwood ◽  
Adam P.C. Smith ◽  
Fiona C. Gardiner ◽  
Rosaria Costa ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Actin Binding ◽  
The Novel ◽  
Cellular Processes ◽  
New Class ◽  
Actin Binding Domain ◽  
Kinetics Of ◽  
Actin Binding Site

Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.

scholarly journals Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association.

1993 ◽  
Vol 121 (3) ◽  
pp. 607-619 ◽  
Author(s):  
G Wiche ◽  
D Gromov ◽  
A Donovan ◽  
M J Castañón ◽  
E Fuchs
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Cultured Cells ◽  
Globular Domain ◽  
Mutant Proteins ◽  
Terminal Domain ◽  
Confocal Immunofluorescence Microscopy ◽  
Terminal Repeats ◽  
Carboxy Terminal

Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.

scholarly journals Cryo-electron microscopy structures of pyrene-labeled ADP-Pi- and ADP-actin filaments

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Steven Z. Chou ◽  
Thomas D. Pollard
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Active Site ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Actin Binding ◽  
Muscle Actin ◽  
Hydrophobic Cavity ◽  
Cryo Electron Microscopy ◽  
Kinetics Of

AbstractSince the fluorescent reagent N-(1-pyrene)iodoacetamide was first used to label skeletal muscle actin in 1981, the pyrene-labeled actin has become the most widely employed tool to measure the kinetics of actin polymerization and the interaction between actin and actin-binding proteins. Here we report high-resolution cryo-electron microscopy structures of actin filaments with N-1-pyrene conjugated to cysteine 374 and either ADP (3.2 Å) or ADP-phosphate (3.0 Å) in the active site. Polymerization buries pyrene in a hydrophobic cavity between subunits along the long-pitch helix with only minor differences in conformation compared with native actin filaments. These structures explain how polymerization increases the fluorescence 20-fold, how myosin and cofilin binding to filaments reduces the fluorescence, and how profilin binding to actin monomers increases the fluorescence.

scholarly journals A33 THE ROLE OF A HOMOZYGOUS PROTEIN CODING VARIANT OF EPS8 IN THE PATHOGENESIS OF PEDIATRIC IBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 40-41
Author(s):  
K Long ◽  
N Warner ◽  
J Pan ◽  
C Guo ◽  
A Muise
Keyword(s):  
Early Onset ◽  
Actin Polymerization ◽  
Cell Structure ◽  
Single Gene ◽  
Actin Binding ◽  
Western Blots ◽  
Very Early Onset Ibd ◽  
Normal Controls ◽  
Pediatric Ibd

Abstract Background The rate of onset for Inflammatory Bowel Disease (IBD) is rising worldwide—most significantly in the pediatric population. The pathogenesis of the disease involves a complicated interaction between the environment and genetics. Recent findings suggest that there is a broad range of rare, single-gene mutations that correlate with the IBD phenotype in children. Some of these single-gene defects can disrupt the epithelial barrier, which alters intestinal immune homeostasis. Epidermal Growth Factor Receptor Kinase Substrate 8 (EPS8) is crucial for the stabilization of F-actin by capping and bundling the barbed end. Previous literature shows that loss of expression of EPS8 disrupts the polymerization of F-actin in intestinal microvilli resulting in shortened brush borders. We identified a pediatric IBD patient with a homozygous missense EPS8 mutation(c.2099C>T, I700T) in the actin-binding domain. The patient presented with pancolitis, colonic strictures and other IBD-like symptoms at 6 weeks of age. Currently, the functional role of EPS8 in the pathogenesis of very early-onset IBD remains elusive. Aims 1)To investigate if the patient with EPS8 mutation has microvilli disorganization. 2)To determine EPS8 localization in patient samples and cell models. 3)To clarify the mechanism of how the EPS8 mutation affects F-actin in vitro. Ultimately, we want to clarify how the mutation of EPS8 might contribute to the onset of pediatric IBD. Methods A rare, damaging mutation in EPS8 was identified in a very early-onset IBD patient by Whole Exome Sequencing. From patient-derived colon biopsy samples, the localization of EPS8 and actin was visualized by immunofluorescence (IF) microscopy. The morphology of the patient’s intestinal microvilli was assessed using transmission electron microscopy (TEM). EPS8 expression was measured using western blot. An F-actin polymerization assay was performed comparing purified WT and mutant EPS8 protein to assess the rate of polymerization from monomeric G-actin to F-actin. Results IF microscopy of the colonic sections revealed lower co-localization of EPS8 and actin on the apical surfaces, compared to IBD and normal controls. Closer examination of the cell structure using TEM showed disruption of the microvilli in the EPS8 mutant, but not in IBD or normal controls. Western blots showed no differences in protein expression between wildtype and mutant EPS8 in HEK293T cells. F-actin polymerization assay revealed differences in the rate of G-actin to F-actin polymerization between wildtype and mutant EPS8. Conclusions The patient with EPS8 mutation had microvilli disorganization. EPS8 localized differently in the patient colon tissues compared to normal and IBD control samples. The EPS8 mutation may affect F-actin polymerization, which may lead to disruption of the microvilli. Funding Agencies CIHR

scholarly journals SUN-213 The Role of Filamin A (FLNA) in the Regulation of Insulin-Like Growth Factor System in Adrenocortical Carcinomas

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Erika Peverelli ◽  
Rosa Catalano ◽  
Elena Giardino ◽  
Donatella Treppiedi ◽  
Federica Mangili ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cultured Cells ◽  
Specific Inhibitor ◽  
Actin Binding ◽  
Endocrine Tumors ◽  
Insulin Like Growth Factor ◽  
Filamin A ◽  
Erk Phosphorylation ◽  
Adrenocortical Carcinomas

Abstract Adrenocortical carcinomas (ACCs) are rare endocrine tumors with poor prognosis. They overexpress insulin-like growth factor 2 (IGF2), that drives a proliferative autocrine loop by binding to IGF1R and IR, with molecular dynamics still poorly identified. Although promising, IGF1R/IR-targeted therapies have demonstrated a limited efficacy in clinical trials in ACC patients. The cytoskeleton actin-binding protein filamin A (FLNA) was shown to impair IR and IGF1R signalling in melanoma and neural progenitor cells, respectively. The aims of this study were to test in ACC cells: 1) FLNA involvement in regulating IGF1R and IR expression and signalling; 2) FLNA role in modulating responsiveness to IGF1R and IGFR/IR inhibitors; 3) FLNA expression in ACCs and correlation with IGF system. In ACC cells we found by immunoprecipitation that both IGF1R and IR interacted with FLNA in basal condition, with an increased or decreased FLNA recruitment to IGF1R and to IR, respectively, after IGF2 stimulation. Genetic silencing of FLNA in ACC cell lines H295R and SW13 induced a significant increase of IGF1R expression (1.4- and 2.3-fold, respectively) and a reduction of IR (-85.5±9.1%, p<0.001 and -32±19.1%, respectively, p<0.05), with a downstream effect of increased cell proliferation (130±13.4%, p<0.01 in H295R and 144.3±23.3%, p<0.01 in SW13 cells) accompanied by an enhanced ERK phosphorylation. Accordingly, in ACC primary cultured cells FLNA silencing increased IGF1R levels (2.9-fold) and enhanced IGF2 effects on ERK phosphorylation by 2.2-fold. In addition, FLNA knockdown potentiated the antiproliferative effects of IGF1R/IR inhibitor Linsitinib and IGF1R specific inhibitor NVP-ADW742 in H295R cells and SW13. This key role of FLNA was even more evident in A7/M2 melanoma cell model, since IGF2 and Linsitinib exerted the expected effects on ERK phosphorylation in M2 cells, lacking FLNA, but not in FLNA-expressing counterpart (A7 cells). Finally, western blot analysis showed significantly lower, although variable, FLNA expression in ACCs (n=10) than in adrenocortical adenomas (ACAs) (n=10) (FLNA/GAPDH ratio 0.37±0.38 and 0.90±0.63, respectively, p<0.05). Interestingly, FLNA/IGF1R ratio inversely correlated with ERK phosphorylation status in ACCs (p<0.05) but not in ACA. In conclusion, we demonstrated that low levels of FLNA enhance both IGF2 proliferative effects and IGF1R/IR inhibitors efficacy in ACC cells, suggesting FLNA as a new factor possibly influencing tumor clinical behavior and the response to the therapy with IGF1R/IR-targeted drugs.

scholarly journals Distinct Roles for CARMIL Isoforms in Cell Migration

2009 ◽  
Vol 20 (24) ◽  
pp. 5290-5305 ◽  
Author(s):  
Yun Liang ◽  
Hanspeter Niederstrasser ◽  
Marc Edwards ◽  
Charles E. Jackson ◽  
John A. Cooper
Keyword(s):  
Cell Migration ◽  
Intermediate Filaments ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
The Other ◽  
Capping Protein ◽  
Myosin I ◽  
Conserved Genes ◽  
Phenotype Expression

Molecular mechanisms for cell migration, especially how signaling and cytoskeletal systems are integrated, are not understood well. Here, we examined the role of CARMIL (capping protein, Arp2/3, and Myosin-I linker) family proteins in migrating cells. Vertebrates express three conserved genes for CARMIL, and we examined the functions of the two CARMIL genes expressed in migrating human cultured cells. Both isoforms, CARMIL1 and 2, were necessary for cell migration, but for different reasons. CARMIL1 localized to lamellipodia and macropinosomes, and loss of its function caused loss of lamellipodial actin, along with defects in protrusion, ruffling, and macropinocytosis. CARMIL1-knockdown cells showed loss of activation of Rac1, and CARMIL1 was biochemically associated with the GEF Trio. CARMIL2, in contrast, colocalized with vimentin intermediate filaments, and loss of its function caused a distinctive multipolar phenotype. Loss of CARMIL2 also caused decreased levels of myosin-IIB, which may contribute to the polarity phenotype. Expression of one CARMIL isoform was not able to rescue the knockdown phenotypes of the other. Thus, the two isoforms are both important for cell migration, but they have distinct functions.

scholarly journals Actin assembly in electropermeabilized neutrophils: role of intracellular calcium.

1990 ◽  
Vol 110 (6) ◽  
pp. 1975-1982 ◽  
Author(s):  
G P Downey ◽  
C K Chan ◽  
S Trudel ◽  
S Grinstein
Keyword(s):  
Actin Polymerization ◽  
Cytosolic Calcium ◽  
Leukotriene B4 ◽  
Human Neutrophils ◽  
Actin Assembly ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Sequence Of Events ◽  
Formyl Peptide

Assembly of microfilaments involves the conversion of actin from the monomeric (G) to the filamentous (F) form. The exact sequence of events responsible for this conversion is yet to be defined and, in particular, the role of calcium remains unclear. Intact and electropermeabilized human neutrophils were used to assess more directly the role of cytosolic calcium [( Ca2+]i) in actin assembly. Staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and right angle light scattering were used to monitor the formation of F-actin. Though addition of Ca2+ ionophores can be known to induce actin assembly, the following observations suggest that an increased [Ca2+]i is not directly responsible for receptor-induced actin polymerization: (a) intact cells in Ca2(+)-free medium, depleted of internal Ca2+ by addition of ionophore, responded to the formyl peptide fMLP with actin assembly despite the absence of changes in [Ca2+]i, assessed with Indo-1; (b) fMLP induced a significant increase in F-actin content in permeabilized cells equilibrated with medium containing 0.1 microM free Ca2+, buffered with up to 10 mM EGTA; (c) increasing [Ca2+]i beyond the resting level by direct addition of CaCl2 to permeabilized cells resulted in actin disassembly. Conversely, lowering [Ca2+]i resulted in spontaneous actin assembly. To reconcile these findings with the actin-polymerizing effects of Ca2+ ionophores, we investigated whether A23187 and ionomycin induced actin assembly by a mechanism independent of, or secondary to the increase in [Ca2+]i. We found that the ionophore-induced actin assembly was completely inhibited by the leukotriene B4 (LTB4) antagonist LY-223982, implying that the ionophore effect was secondary to LTB4 formation, possibly by stimulation of phospholipase A2. We conclude that actin assembly is not mediated by an increase in [Ca2+]i, but rather that elevated [Ca2+]i facilitates actin disassembly, an effect possibly mediated by Ca2(+)-sensitive actin filament-severing proteins such as gelsolin. Sequential actin assembly and disassembly may be necessary for functions such as chemotaxis.

scholarly journals Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein.

1988 ◽  
Vol 107 (4) ◽  
pp. 1489-1498 ◽  
Author(s):  
T Ankenbauer ◽  
J A Kleinschmidt ◽  
J Vandekerckhove ◽  
W W Franke
Keyword(s):  
Xenopus Laevis ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Early Embryogenesis ◽  
Actin Binding ◽  
Dependent Manner ◽  
Expression Library ◽  
Cytoplasmic Actin ◽  
Carboxy Terminal

Oocytes, notably those of amphibia, accumulate large pools of nonfilamentous ("soluble") actin, both in the cytoplasm and in the nucleoplasm, which coexist with extensive actin filament arrays in the cytoplasmic cortex. Because the regulation of oogenically accumulated actin is important in various processes of oogenesis, egg formation, fertilization and early embryogenesis, we have purified and characterized the major actin-binding proteins present in oocytes of Xenopus laevis. Here we report that the major actin-binding component in the ooplasm, but not in the nucleus, is a polypeptide of Mr approximately 93,000 on SDS-PAGE that reduces actin polymerization in vitro in a Ca2+-dependent manner but promotes nucleation events, and also reduces the viscosity of actin polymers, indicative of severing activity. We have raised antibodies against the purified oocyte protein and show that it is different from villin, is also prominent in unfertilized eggs and early embryos and is very similar to a corresponding protein present in various tissues and in cultured cells, and appears to be spread over the cytoplasm. Using these antibodies we have isolated a cDNA clone from a lambda gt11 expression library of ovarian poly(A)+-RNA. Determination of the amino acid sequence derived from the nucleotide sequence, together with the directly determined sequence of the amino terminus of the native protein, has shown that this clone encodes the carboxy-terminal half of gelsolin. We conclude that gelsolin is the major actin-modulating protein in oogenesis and early embryogenesis of amphibia, and probably also of other species, that probably also plays an important role in the various Ca2+-dependent gelation and contractility processes characteristic of these development stages.

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