scholarly journals Analysis of Unregulated Formin Activity Reveals How Yeast Can Balance F-Actin Assembly between Different Microfilament-based Organizations

2008 ◽  
Vol 19 (4) ◽  
pp. 1474-1484 ◽  
Author(s):  
Lina Gao ◽  
Anthony Bretscher
Keyword(s):  
Cdna Library ◽  
Actin Filament ◽  
Actin Binding ◽  
Actin Assembly ◽  
The Third ◽  
Filament Assembly ◽  
Actin Cables ◽  
Assembly Pathways ◽  
Massive Accumulation ◽  
Proper Balance

Formins are regulated actin-nucleating proteins that are widespread among eukaryotes. Overexpression of unregulated formins in budding yeast is lethal and causes a massive accumulation of disorganized cable-like filaments. To explore the basis of this lethality, a cDNA library was screened to identify proteins whose overexpression could rescue the lethality conferred by unregulated Bnr1p expression. Three classes of suppressors encoding actin-binding proteins were isolated. One class encodes proteins that promote the assembly of actin cables (TPM1, TPM2, and ABP140), suggesting that the lethality was rescued by turning disorganized filaments into functional cables. The second class encodes proteins that bind G-actin (COF1, SRV2, and PFY1), indicating that reduction of the pool of actin available for cable formation may also rescue lethality. Consistent with this, pharmacological or genetic reduction of available actin also protected the cell from overproduction of unregulated Bnr1p. The third class consists of Las17p, an activator of the formin-independent Arp2/3p-dependent actin nucleation pathway. These results indicate that proper assembly of actin cables is sensitive to the appropriate balance of their constituents and that input into one pathway for actin filament assembly can affect another. Thus, cells must have a way of ensuring a proper balance between actin assembly pathways.

scholarly journals LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

2018 ◽  
Author(s):  
Xiaohua Hu ◽  
R. Dyche Mullins
Keyword(s):  
Actin Filament ◽  
Network Formation ◽  
De Novo ◽  
Actin Binding ◽  
Actin Assembly ◽  
Autophagosome Formation ◽  
Actin Networks ◽  
Filament Assembly ◽  
Filament Networks ◽  
Actin Comet Tails

AbstractDuring autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic JMY co-localizes with STRAP, a regulator of JMY’s nuclear functions, on non-motile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY’s de novo actin nucleation activity via a cryptic actin-binding sequence near JMY’s N-terminus, and STRAP inhibits JMY’s ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3‐ and JMY-dependent actin network formation on membranes, and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY’s actin assembly activities in trans during autophagy.eTOC BlurbThe actin regulator JMY creates filament networks that move membranes during autophagy. We find that, in unstarved cells, JMY is inhibited by interaction with the STRAP protein, but upon starvation JMY is recruited away from STRAP and activated by LC3.

scholarly journals LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation

2018 ◽  
Vol 218 (1) ◽  
pp. 251-266 ◽  
Author(s):  
Xiaohua Hu ◽  
R. Dyche Mullins
Keyword(s):  
Actin Filament ◽  
Network Formation ◽  
De Novo ◽  
Actin Binding ◽  
Actin Assembly ◽  
Autophagosome Formation ◽  
Actin Networks ◽  
Filament Assembly ◽  
Filament Networks ◽  
Actin Comet Tails

During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic JMY colocalizes with STRAP, a regulator of JMY’s nuclear functions, on nonmotile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY’s de novo actin nucleation activity via a cryptic actin-binding sequence near JMY’s N terminus, and STRAP inhibits JMY’s ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY’s actin assembly activities in trans during autophagy.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

scholarly journals The actin released from profilin--actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes.

1990 ◽  
Vol 110 (6) ◽  
pp. 1965-1973 ◽  
Author(s):  
F S Southwick ◽  
C L Young
Keyword(s):  
Actin Filament ◽  
Polymorphonuclear Leukocytes ◽  
Critical Concentration ◽  
Relative Concentration ◽  
Insoluble Fraction ◽  
Membrane Receptors ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Total Change ◽  
Filament Assembly

Chemoattractant stimulation of polymorphonuclear leukocytes is associated with a nearly two-fold rise in actin filament content. We examined the role of the actin monomer sequestering protein, profilin, in the regulation of PMN actin filament assembly during chemoattractant stimulation using a Triton extraction method. Poly-L-proline-conjugated Sepharose beads were used to assess the relative concentration of actin bound to profilin with high enough affinity to withstand dilution (profilin-actin complex) and DNase I-conjugated beads to measure the relative concentration of actin in the Triton-soluble fraction not bound to profilin. Actin associated with the Triton-insoluble fraction (F-actin) was also measured. In unstimulated PMN, the relative concentration of actin bound to profilin was maximum. After FMLP stimulation, profilin released actin monomers within 10 s, with the profilin-actin complex concentration reaching a nadir by 40 s and remaining low as long as the cells were exposed to chemoattractant (up to 30 min). If FMLP was dissociated from PMN membrane receptors using t-BOC, actin reassociated with profilin within 20 s. Quantitative analysis of these reactions, however, revealed that profilin release of and rebinding to actin could account for only a small percentage of the total change in F-actin content. Determination of the total profilin and actin concentrations in PMN revealed that the molar ratio of profilin to actin was 1 to 5.2. When purified actin was polymerized in PMN Triton extract containing EGTA, removal of profilin from the extract minimally affected (12% reduction) the high apparent critical concentration at which actin began to assemble. Although profilin released actin at the appropriate time to stimulate actin assembly during exposure to chemoattractants, the concentration of profilin in PMN was insufficient to explain the high unpolymerized actin content in unstimulated PMN and the quantity of actin released from profilin too small to account for the large shifts from unpolymerized to polymerized actin associated with maximal chemoattractant stimulation.

Actin Assembly Takes Off Like a Rocket!

2013 ◽  
Vol 21 (2) ◽  
pp. 8-9
Author(s):  
Stephen W. Carmichael ◽  
Jeffrey L. Salisbury
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cells ◽  
Actin Assembly ◽  
Elongation Factors ◽  
Delicate Balance ◽  
Filament Assembly ◽  
Two Factors

Actin filament assembly occurs in all eukaryotic cells and involves a delicate balance between factors that promote assembly and factors that inhibit assembly. Filament assembly begins with a process of nucleation and then proceeds via elongation. Filament assembly in vivo requires nucleation and elongation factors to overcome barriers that could either bind actin monomers to inhibit nucleation or “cap” the ends of elongating filaments. The formation of most cellular actin structures depends on two or more such factors, which may interact directly. The interaction between two factors that initiate nucleation and promote assembly has recently been demonstrated by Dennis Breitsprecher, Richa Jaiswal, Jeffrey Bombardier, Christopher Gould, Jeff Gelles, and Bruce Goode. Interestingly, the model of these factors in action (Figure 1) resembles a rocket launcher!

scholarly journals Post-mitotic expansion of cell nuclei requires ACTN4-mediated nuclear actin filament bundling

2020 ◽  
Author(s):  
Sylvia Krippner ◽  
Jannik Winkelmeier ◽  
Carsten Schwan ◽  
Julian Knerr ◽  
David Virant ◽  
...  
Keyword(s):  
Actin Filament ◽  
Super Resolution ◽  
Mitotic Cell ◽  
Actin Binding ◽  
Nuclear Actin ◽  
Actin Assembly ◽  
Cell Nuclei ◽  
Cellular Processes ◽  
And Migration ◽  
Nuclear Expansion

AbstractThe actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, as well as membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle ACTN4 as a critical regulator to facilitate F-actin formation, reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filaments and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei we find that ACTN4 is necessary for postmitotic nuclear actin assembly and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size during mitotic cell division.

scholarly journals Inhibition of CapZ during myofibrillogenesis alters assembly of actin filaments.

1995 ◽  
Vol 128 (1) ◽  
pp. 61-70 ◽  
Author(s):  
D A Schafer ◽  
C Hug ◽  
J A Cooper
Keyword(s):  
Monoclonal Antibody ◽  
Actin Filament ◽  
Actin Filaments ◽  
Binding Activity ◽  
Actin Binding ◽  
Mutant Form ◽  
Actin Binding Protein ◽  
Filament Assembly ◽  
Skeletal Myotubes ◽  
Aligned Array

The actin filaments of myofibrils are highly organized; they are of a uniform length and polarity and are situated in the sarcomere in an aligned array. We hypothesized that the barbed-end actin-binding protein, CapZ, directs the process of actin filament assembly during myofibrillogenesis. We tested this hypothesis by inhibiting the actin-binding activity of CapZ in developing myotubes in culture using two different methods. First, injection of a monoclonal antibody that prevents the interaction of CapZ and actin disrupts the non-striated bundles of actin filaments formed during the early stages of myofibril formation in skeletal myotubes in culture. The antibody, when injected at concentrations lower than that required for disrupting the actin filaments, binds at nascent Z-disks. Since the interaction of CapZ and the monoclonal antibody are mutually exclusive, this result indicates that CapZ binds nascent Z-disks independent of an interaction with actin filaments. In a second approach, expression in myotubes of a mutant form of CapZ that does not bind actin results in a delay in the appearance of actin in a striated pattern in myofibrils. The organization of alpha-actinin at Z-disks also is delayed, but the organization of titin and myosin in sarcomeres is not significantly altered. We conclude that the interaction of CapZ and actin is important for the organization of actin filaments of the sarcomere.

An Aplysia cell adhesion molecule associated with site-directed actin filament assembly in neuronal growth cones

1996 ◽  
Vol 109 (12) ◽  
pp. 2843-2854 ◽  
Author(s):  
C. Thompson ◽  
C.H. Lin ◽  
P. Forscher
Keyword(s):  
Cell Adhesion ◽  
Membrane Protein ◽  
Actin Filament ◽  
De Novo ◽  
Lateral Diffusion ◽  
Actin Assembly ◽  
Neuronal Growth ◽  
Cytoskeletal Remodeling ◽  
Interaction Sites ◽  
Filament Assembly

During neuronal growth cone-target interactions, a programmed sequence of cytoskeletal remodeling has been described, involving increased actin assembly at the target site and directed microtubule extension into it. The cell adhesion protein apCAM rapidly accumulates at such interaction sites, suggesting a possible role in regulating cytoskeletal remodeling. To test this hypothesis we crosslinked apCAM to varying degrees with antibodies. Secondary immunocomplexes exhibited a classical patching and capping response; in contrast, high density crosslinking of apCAM by antibody coated beads triggered localized actin assembly accompanied by formation of tail-like actin structures referred to as inductopodia. When beads were derivatized with increasing amounts of anti-apCAM they displayed three sequential dose-dependent kinetic states after binding: (1) lateral diffusion in the plane of the membrane; (2) restricted diffusion due to coupling with underlying F-actin; and (3) translocation in the plane of the membrane driven by de novo actin filament assembly local to bead binding sites, i.e. inductopodia formation. In contrast, lectin coated beads were far less efficient in triggering inductopodia formation despite demonstrated membrane protein binding. This work provides evidence that crosslinking of a diffusable membrane protein, apCAM, to threshold levels, can trigger highly localized actin filament assembly and rapid remodeling of neuronal cytoarchitecture.

The relationship between CR3 deficiency and neutrophil actin assembly

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1973-1979
Author(s):  
FS Southwick ◽  
TH Howard ◽  
T Holbrook ◽  
DC Anderson ◽  
TP Stossel ◽  
...  
Keyword(s):  
Actin Filament ◽  
Polymorphonuclear Leukocytes ◽  
Family Members ◽  
Surface Expression ◽  
Actin Assembly ◽  
Normal Surface ◽  
Filament Assembly ◽  
Type 3 ◽  
The Relationship ◽  
Total Concentrations

Polymorphonuclear leukocytes (PMN) with a deficiency of the complement receptor type 3 (CR3) membrane glycoprotein family have impairments in the ability to adhere to surfaces as well as chemotactic and phagocytic defects, processes that require a functional contractile apparatus. PMN from the patient with neutrophil actin dysfunction (NAD) displayed similar functional characteristics to those with CR3 deficiency suggesting the two disorders may be the same disease. In order to evaluate the relationship between CR3 deficiency and actin assembly, actin filament assembly was measured in PMN from six previously reported homozygotes (two severe and four moderate CR3-deficient patients) as well as five heterozygotes for CR3 deficiency. PMN from all patients had normal unstimulated concentrations of F-actin and after exposure to the chemotactic peptide FMLP (5 x 10(-7) mol/L for 5 to 40 seconds at 25 degrees C) assembled actin normally. Pretreatment of normal PMN with concentrations of monoclonal anti-alpha CR3 antibody, capable of blocking PMN adherence, also failed to impair FMLP- induced actin filament assembly. CR3 glycoprotein expression was measured in PMNs from the mother, father, and older sister of the NAD patient (N Engl J Med 291:1093, 1974). Actin filament assembly was recently shown to be defective in PMNs from all three family members. The total concentrations of the alpha and beta CR3 subunits were below normal in PMN detergent extracts from the mother (25% of simultaneous controls) and older sister (56% of control). PMN surface expression of these two subunits was also found to be depressed (mother, 50%; older sister, 63% of control). These findings suggest these two NAD family members are heterozygote carriers for CR3 deficiency as well as NAD. Simultaneous studies of the father, however, demonstrated normal total concentrations of both the alpha and beta CR3 subunits (126% of controls) as well as normal surface expression of both subunits after phorbol myristate acetate stimulation and incubation at 37 degrees C (mean, 112% of controls) but slightly lower than normal levels after FMLP stimulation (mean, 83%). These findings indicate that CR3 deficiency generally is not associated with defective actin filament assembly and support the conclusion that NAD represents a unique kindred in which PMN actin function differs from previously reported genotypes of CR3 deficiency.

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