scholarly journals The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase

2020 ◽  
Vol 6 (40) ◽  
pp. eabb7854 ◽  
Author(s):  
Riyad N. H. Seervai ◽  
Rahul K. Jangid ◽  
Menuka Karki ◽  
Durga Nand Tripathi ◽  
Sung Yun Jung ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Set Domain ◽  
Interacting Protein ◽  
Lysine Methyltransferase ◽  
Huntingtin Interacting Protein ◽  
In Cells

The methyltransferase SET domain–containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals HYPK scaffolds the Nedd8 and LC3 proteins to initiate the formation of autophagosome around the poly-neddylated huntingtin exon1 aggregates

2019 ◽  
Author(s):  
Debasish Kumar Ghosh ◽  
Ajit Roy ◽  
Akash Ranjan
Keyword(s):  
Regulatory Proteins ◽  
Scaffolding Protein ◽  
Cell Physiology ◽  
Autophagy Flux ◽  
Interacting Protein ◽  
Cellular Autophagy ◽  
Uba Domain ◽  
Autophagic Degradation ◽  
Huntingtin Interacting Protein ◽  
Conserved Amino Acids

ABSTRACTSelective autophagy of protein aggregates is necessary for maintaining the cellular proteostasis. Several regulatory proteins play critical roles in this process. Here, we report that the huntingtin interacting protein K (HYPK) modulates the autophagic degradation of poly-neddylated huntingtin exon1 aggregates. HYPK functions as a scaffolding protein that binds to the Nedd8 and LC3 proteins. The C-terminal ubiquitin-associated (UBA) domain of HYPK binds to the Nedd8, whereas an N-terminal tyrosine-type (Y-type) LC3 interacting region (LIR) of HYPK binds to the LC3. Several conserved amino acids in the UBA domain of HYPK are necessary to mediate the efficient binding of HYPK to Nedd8. The autophagy inducing properties of HYPK are manifested by the increased lipidation of LC3 protein, increased expression of beclin-1 and ATG-5 proteins, and generation of puncta-like granules of LC3 in the HYPK overexpressing cells. Association of the ‘H-granules’ of HYPK with the poly-neddylated huntingtin exon1 aggregates results in the formation of autophagosome around the huntingtin exon1 aggregates, thereby clearing the aggregates by aggrephagy. Poly-neddylation of huntingtin exon1 is required for its autophagic degradation by HYPK. Thus, overexpression of Nedd8 also increases the basal level of cellular autophagy, other than maintaining the autophagy flux. The poly-neddylation dependent autophagic clearance of huntingtin exon1 by HYPK leads to better cell physiology and survival. Taken together, our study describes a novel mechanism of HYPK mediated autophagy of poly-neddylated huntingtin exon1 aggregates.

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 Actin-depolymerizing Factor and Cofilin-1 Play Overlapping Roles in Promoting Rapid F-Actin Depolymerization in Mammalian Nonmuscle Cells

2005 ◽  
Vol 16 (2) ◽  
pp. 649-664 ◽  
Author(s):  
Pirta Hotulainen ◽  
Eija Paunola ◽  
Maria K. Vartiainen ◽  
Pekka Lappalainen
Keyword(s):  
Cell Motility ◽  
Actin Filament ◽  
Actin Filaments ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Actin Depolymerizing Factor ◽  
Cytoskeletal Dynamics ◽  
Nonmuscle Cells ◽  
In Cells

Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling “old” actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals The PCH Family Member Proline-Serine-Threonine Phosphatase–interacting Protein 1 Targets to the Leukocyte Uropod and Regulates Directed Cell Migration

2008 ◽  
Vol 19 (8) ◽  
pp. 3180-3191 ◽  
Author(s):  
Kate M. Cooper ◽  
David A. Bennin ◽  
Anna Huttenlocher
Keyword(s):  
Cell Migration ◽  
Family Member ◽  
Actin Polymerization ◽  
Stable Population ◽  
Type I ◽  
Microtubule Network ◽  
Interacting Protein ◽  
Directed Cell Migration ◽  
Transferrin Uptake ◽  
Dynamin 2

Pombe Cdc15 homology (PCH) family members have emerged as important regulators of membrane–cytoskeletal interactions. Here we show that PSTPIP1, a PCH family member expressed in hematopoietic cells, regulates the motility of neutrophil-like cells and is a novel component of the leukocyte uropod where it colocalizes with other uropod components, such as type I PIPKIγ. Furthermore, we show that PSTPIP1 association with the regulator of endocytosis, dynamin 2, and PSTPIP1 expression impairs transferrin uptake and endocytosis. We also show that PSTPIP1 localizes at the rear of neutrophils with a subpopulation of F-actin that is specifically detected by the binding of an F-actin probe that detects a more stable population of actin. Finally, we show that actin polymerization, but not the microtubule network, is necessary for the polarized distribution of PSTPIP1 toward the rear of the cell. Together, our findings demonstrate that PSTPIP1 is a novel component of the leukocyte uropod that regulates endocytosis and cell migration.

scholarly journals The integral membrane protein, ponticulin, acts as a monomer in nucleating actin assembly.

1993 ◽  
Vol 120 (4) ◽  
pp. 909-922 ◽  
Author(s):  
C P Chia ◽  
A Shariff ◽  
S A Savage ◽  
E J Luna
Keyword(s):  
Membrane Protein ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Lipid Vesicles ◽  
Integral Membrane Protein ◽  
Actin Binding ◽  
Actin Assembly ◽  
Nucleation Activity

Ponticulin, an F-actin binding transmembrane glycoprotein in Dictyostelium plasma membranes, was isolated by detergent extraction from cytoskeletons and purified to homogeneity. Ponticulin is an abundant membrane protein, averaging approximately 10(6) copies/cell, with an estimated surface density of approximately 300 per microns2. Ponticulin solubilized in octylglucoside exhibited hydrodynamic properties consistent with a ponticulin monomer in a spherical or slightly ellipsoidal detergent micelle with a total molecular mass of 56 +/- 6 kD. Purified ponticulin nucleated actin polymerization when reconstituted into Dictyostelium lipid vesicles, but not when a number of commercially available lipids and lipid mixtures were substituted for the endogenous lipid. The specific activity was consistent with that expected for a protein comprising 0.7 +/- 0.4%, by mass, of the plasma membrane protein. Ponticulin in octylglucoside micelles bound F-actin but did not nucleate actin assembly. Thus, ponticulin-mediated nucleation activity was sensitive to the lipid environment, a result frequently observed with transmembrane proteins. At most concentrations of Dictyostelium lipid, nucleation activity increased linearly with increasing amounts of ponticulin, suggesting that the nucleating species is a ponticulin monomer. Consistent with previous observations of lateral interactions between actin filaments and Dictyostelium plasma membranes, both ends of ponticulin-nucleated actin filaments appeared to be free for monomer assembly and disassembly. Our results indicate that ponticulin is a major membrane protein in Dictyostelium and that, in the proper lipid matrix, it is sufficient for lateral nucleation of actin assembly. To date, ponticulin is the only integral membrane protein known to directly nucleate actin polymerization.

scholarly journals Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

2008 ◽  
Vol 182 (2) ◽  
pp. 341-353 ◽  
Author(s):  
Hao Yuan Kueh ◽  
Guillaume T. Charras ◽  
Timothy J. Mitchison ◽  
William M. Brieher
Keyword(s):  
Actin Filaments ◽  
Mammalian Cells ◽  
Cytochalasin D ◽  
Reaction Intermediates ◽  
Latrunculin B ◽  
Interacting Protein ◽  
Physiological Relevance ◽  
High Concentrations ◽  
End Capping ◽  
In Cells

Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs.

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