Tropomyosin-Based Regulation of the Actin Cytoskeleton in Time and Space

2008 ◽  
Vol 88 (1) ◽  
pp. 1-35 ◽  
Author(s):  
Peter Gunning ◽  
Geraldine O’neill ◽  
Edna Hardeman
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Cell Types ◽  
Actin Binding ◽  
Regulation Of Expression ◽  
Wide Range ◽  
Muscle Tropomyosin ◽  
The Many

Tropomyosins are rodlike coiled coil dimers that form continuous polymers along the major groove of most actin filaments. In striated muscle, tropomyosin regulates the actin-myosin interaction and, hence, contraction of muscle. Tropomyosin also contributes to most, if not all, functions of the actin cytoskeleton, and its role is essential for the viability of a wide range of organisms. The ability of tropomyosin to contribute to the many functions of the actin cytoskeleton is related to the temporal and spatial regulation of expression of tropomyosin isoforms. Qualitative and quantitative changes in tropomyosin isoform expression accompany morphogenesis in a range of cell types. The isoforms are segregated to different intracellular pools of actin filaments and confer different properties to these filaments. Mutations in tropomyosins are directly involved in cardiac and skeletal muscle diseases. Alterations in tropomyosin expression directly contribute to the growth and spread of cancer. The functional specificity of tropomyosins is related to the collaborative interactions of the isoforms with different actin binding proteins such as cofilin, gelsolin, Arp 2/3, myosin, caldesmon, and tropomodulin. It is proposed that local changes in signaling activity may be sufficient to drive the assembly of isoform-specific complexes at different intracellular sites.

NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

2002 ◽  
Vol 115 (15) ◽  
pp. 3207-3222 ◽  
Author(s):  
Yen-Yi Zhen ◽  
Thorsten Libotte ◽  
Martina Munck ◽  
Angelika A. Noegel ◽  
Elena Korenbaum
Keyword(s):  
Actin Cytoskeleton ◽  
Transmembrane Domain ◽  
Coiled Coil ◽  
Peripheral Blood Lymphocytes ◽  
Binding Domain ◽  
Actin Binding ◽  
Actin Binding Domain ◽  
Microfilament System

NUANCE (NUcleus and ActiN Connecting Element) was identified as a novel protein with an α-actinin-like actin-binding domain. A human 21.8 kb cDNA of NUANCE spreads over 373 kb on chromosome 14q22.1-q22.3. The cDNA sequence predicts a 796 kDa protein with an N-terminal actin-binding domain, a central coiled-coil rod domain and a predicted C-terminal transmembrane domain. High levels of NUANCE mRNA were detected in the kidney, liver,stomach, placenta, spleen, lymphatic nodes and peripheral blood lymphocytes. At the subcellular level NUANCE is present predominantly at the outer nuclear membrane and in the nucleoplasm. Domain analysis shows that the actin-binding domain binds to Factin in vitro and colocalizes with the actin cytoskeleton in vivo as a GFP-fusion protein. The C-terminal transmembrane domain is responsible for the targeting the nuclear envelope. Thus, NUANCE is the firstα-actinin-related protein that has the potential to link the microfilament system with the nucleus.

Nuclear ion channel activity is regulated by actin filaments

1996 ◽  
Vol 270 (5) ◽  
pp. C1532-C1543 ◽  
Author(s):  
A. G. Prat ◽  
H. F. Cantiello
Keyword(s):  
Ion Channels ◽  
Actin Cytoskeleton ◽  
Nuclear Envelope ◽  
Ion Channel ◽  
Actin Filaments ◽  
Actin Binding ◽  
Bathing Solution ◽  
Channel Activity ◽  
Nuclear Ion Channels ◽  
Inside Out

Actin filaments are novel second messengers involved in ion channel regulation. Because cytoskeletal components interact with the nuclear envelope, the actin cytoskeleton may also control nuclear membrane function. In this report, the patch-clamp technique was applied to isolated nuclei from amphibian A6 epithelial cells to assess the role of actin filaments on nuclear ion channel activity under nucleus-attached or -excised conditions. The most prevalent spontaneous nuclear ion channel species, 76% (n = 46), was cation selective and had a maximal single-channel conductance of approximately 420 pS. Nuclear ion channels also displayed multiple subconductance states, including channel activity of 26 pS that was frequently observed. Nuclear ion channel activity on otherwise quiescent patches was induced by either addition of the actin cytoskeleton disrupter cytochalasin D (CD; 5 micrograms/ml, 60%, 3 of 5 patches) or actin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches (59%, 13 of 22 patches). Actin also induced ion channel activity in quiescent excised inside-out patches from the nuclear envelope (80%, 4 of 5 patches). In contrast, addition of bovine serum albumin (10-1,000 micrograms/ml) to the bathing solution of nucleus-attached patches was without effect on nuclear ion channel activity (5 of 5 patches). The monoclonal antibody MAb414, specific for nuclear pore complex proteins, completely prevented either spontaneous or cytosolic actin-induced nuclear ion channels under nucleus-attached conditions (4 of 4 patches) but not intranuclear actin-induced nuclear ion channels under excised inside-out conditions (3 of 3 patches). In nucleus-attached patches, channel activity was readily activated by addition of the G-actin-binding protein deoxyribonuclease I to nucleus-attached patches (56%, 5 of 9 patches) or further addition of the actin-cross-linker filamin in the presence of actin (57%, 4 of 7 patches). The data indicate that dynamic changes in actin filament organization may represent a novel mechanism to control nuclear function.

Understanding Biological Structures Through Exploring the Mechanical Response of Cell-Like Systems

2008 ◽  
Author(s):  
Ying Zhang ◽  
Philip R. LeDuc
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Filaments ◽  
Living Cell ◽  
Cellular Response ◽  
Cancer Metastasis ◽  
Cell Structure ◽  
Polymer Physics ◽  
Wide Range

The actin cytoskeleton provides mechanical support for the cell and influences activities such as cancer metastasis and chemotaxis. While their mechanical responses have been studied in vivo and in vitro, understanding the link between these two forms remains challenging. To explore this gap and further understand cell structure, we reconstructed the cell cytoskeleton in a membrane-like spherical liposome to mimic the cellular environment; this enables an artificial “cell like” system. Through this approach, we are pursuing a path to compare in vitro mechanics from a polymer physics perspective of individual actin filaments with the in vivo mechanics of a living cell [1]. A living cell contains many organelles, which are in a highly packed environment and require significant organization to function. The actin cytoskeleton provides both structural and organizational regulation that is essential for cellular response. Here, we first encapsulated G-actin into giant unilamellar vesicles through an electroformation technique and then polymerized them into actin filaments (F-actin) within individual vesicles. To probe their conformation, we visualized these vesicles with fluorescence and laser scanning confocal microscopy. We then used a tapping mode atomic force microscopy to determine the mechanical properties of these cell-like systems. These results provide insight into a wide range of fields and studies including polymer physics, cell biology, and biotechnology.

scholarly journals Focal segmental glomerulosclerosis ACTN4 mutants binding to actin: regulation by phosphomimetic mutations

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hanshuang Shao ◽  
Bentley Wingert ◽  
Astrid Weins ◽  
Martin R. Pollak ◽  
Carlos Camacho ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Focal Segmental Glomerulosclerosis ◽  
Actin Filaments ◽  
Binding Activity ◽  
Binding Domain ◽  
Actin Binding ◽  
Dominant Form ◽  
Cell Functions ◽  
Segmental Glomerulosclerosis ◽  
Actin Binding Domain

Abstract Natural mutations such as lysine 255 to glutamic acid (K to E), threonine 259 to isoleucine (T to I) and serine 262 to proline (S to P) that occur within the actin binding domain of alpha-actinin-4 (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) in affected humans. This appears due to elevated actin binding propensity in podocytes resulting in a ‘frozen’ cytoskeleton. What is challenging is how this cellular behavior would be compatible with other cell functions that rely on cytoskeleton plasticity. Our previous finding revealed that wild type ACTN4 can be phosphorylated at tyrosine 4 and 31 upon stimulation by epidermal growth factor (EGF) to reduce the binding to actin cytoskeleton. We queried whether the elevated actin binding activity of FSGS mutants can be downregulated by EGF-mediated phosphorylation, to discern a mechanism by which the actin-cytoskeleton can be released in FSGS. In this manuscript, we first constructed variants with Y4/31E to mimic the phosphorylation at tyrosines 4 and 31 based on earlier modeling simulations that predicted that this would bury the actin binding domains and lead to a decrease in actin binding activity. We found that Y4/31E significantly reduced the actin binding activity of K255E, T259I and S262P, dramatically preventing them from aggregating in, and inhibiting motility of, podocytes, fibroblasts and melanoma cells. A putative kinase target site at Y265 in the actin binding domain was also generated as a phosphomimetic ACTN4 Y265E that demonstrated even greater binding to actin filaments than K255E and the other FSGS mutants. That the tyrosine kinase regulation of FSGS mutation binding to actin filaments can occur in cells was shown by phosphorylation on Y4 and Y31 of the K225E after extended exposure of cells to EGF, with a decrease in ACTN4 aggregates in fibroblasts. These findings will provide evidence for targeting the N-termini of FSGS ACTN4 mutants to downregulate their actin binding activities for ameliorating the glomerulosclerotic phenotype of patients.

scholarly journals Ubiquitination and Long Non-coding RNAs Regulate Actin Cytoskeleton Regulators in Cancer Progression

2019 ◽  
Vol 20 (12) ◽  
pp. 2997 ◽  
Author(s):  
Xuda Ma ◽  
Yamei Dang ◽  
Xiaowen Shao ◽  
Xuechun Chen ◽  
Fei Wu ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cancer Progression ◽  
Rho Gtpases ◽  
Actin Filaments ◽  
Cancer Metastasis ◽  
Coiled Coil ◽  
Lim Domain ◽  
E3 Ligases ◽  
Protein Levels ◽  
Family Protein

Actin filaments are a major component of the cytoskeleton in eukaryotic cells and play an important role in cancer metastasis. Dynamics and reorganization of actin filaments are regulated by numerous regulators, including Rho GTPases, PAKs (p21-activated kinases), ROCKs (Rho-associated coiled-coil containing kinases), LIMKs (LIM domain kinases), and SSH1 (slingshot family protein phosphate 1). Ubiquitination, as a ubiquitous post-transcriptional modification, deceases protein levels of actin cytoskeleton regulatory factors and thereby modulates the actin cytoskeleton. There is increasing evidence showing cytoskeleton regulation by long noncoding RNAs (lncRNAs) in cancer metastasis. However, which E3 ligases are activated for the ubiquitination of actin-cytoskeleton regulators involved in tumor metastasis remains to be fully elucidated. Moreover, it is not clear how lncRNAs influence the expression of actin cytoskeleton regulators. Here, we summarize physiological and pathological mechanisms of lncRNAs and ubiquitination control mediators of actin cytoskeleton regulators which that are involved in tumorigenesis and tumor progression. Finally, we briefly discuss crosstalk between ubiquitination and lncRNA control mediators of actin-cytoskeleton regulators in cancer.

scholarly journals MAGI-1 interacts with Slo1 channel proteins and suppresses Slo1 expression on the cell surface

2009 ◽  
Vol 297 (1) ◽  
pp. C55-C65 ◽  
Author(s):  
Lon D. Ridgway ◽  
Eun Young Kim ◽  
Stuart E. Dryer
Keyword(s):  
Cell Surface ◽  
Outward Current ◽  
Cell Types ◽  
Surface Expression ◽  
Scaffolding Protein ◽  
Actin Binding ◽  
Differentiated Cells ◽  
Cell Surface Biotinylation ◽  
Bait Construct ◽  
Wide Range

Large conductance Ca2+-activated K+ (BKCa) channels encoded by the Slo1 gene (also known as KCNMA1) are physiologically important in a wide range of cell types and form complexes with a number of other proteins that affect their function. We performed a yeast two-hybrid screen to identify proteins that interact with BKCa channels using a bait construct derived from domains in the extreme COOH-terminus of Slo1. A protein known as membrane-associated guanylate kinase with inverted orientation protein-1 (MAGI-1) was identified in this screen. MAGI-1 is a scaffolding protein that allows formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. MAGI-1 is expressed in a number of tissues, including podocytes and the brain. The interaction between MAGI-1 and BKCa channels was confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays in differentiated cells of a podocyte cell line and in human embryonic kidneys (HEK)293T cells transiently coexpressing MAGI-1a and three different COOH-terminal Slo1 variants. Coexpression of MAGI-1 with Slo1 channels in HEK-293T cells results in a significant reduction in the surface expression of Slo1, as assessed by cell-surface biotinylation assays, confocal microscopy, and whole cell recordings. Partial knockdown of endogenous MAGI-1 expression by small interfering RNA (siRNA) in differentiated podocytes increased the surface expression of endogenous Slo1 as assessed by electrophysiology and cell-surface biotinylation assays, whereas overexpression of MAGI-1a reduced steady-state voltage-evoked outward current through podocyte BKCa channels. These data suggest that MAGI-1 plays a role in regulation of surface expression of BKCa channels in the kidney and possibly in other tissues.

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 Comparison of actin and cell surface dynamics in motile fibroblasts.

1992 ◽  
Vol 119 (2) ◽  
pp. 367-377 ◽  
Author(s):  
J A Theriot ◽  
T J Mitchison
Keyword(s):  
Actin Cytoskeleton ◽  
Dynamic Behavior ◽  
Actin Filaments ◽  
Half Life ◽  
Dorsal Surface ◽  
Cell Types ◽  
3T3 Cells ◽  
3T3 Fibroblasts ◽  
Continuous Filament ◽  
Time Required

We have investigated the dynamic behavior of actin in fibroblast lamellipodia using photoactivation of fluorescence. Activated regions of caged resorufin (CR)-labeled actin in lamellipodia of IMR 90 and MC7 3T3 fibroblasts were observed to move centripetally over time. Thus in these cells, actin filaments move centripetally relative to the substrate. Rates were characteristic for each cell type; 0.66 +/- 0.27 microns/min in IMR 90 and 0.36 +/- 0.16 microns/min in MC7 3T3 cells. In neither case was there any correlation between the rate of actin movement and the rate of lamellipodial protrusion. The half-life of the activated CR-actin filaments was approximately 1 min in IMR 90 lamellipodia, and approximately 3 min in MC7 3T3 lamellipodia. Thus continuous filament turnover accompanies centripetal movement. In both cell types, the length of time required for a section of the actin meshwork to traverse the lamellipodium was several times longer than the filament half-life. The dynamic behavior of the dorsal surface of the cell was also observed by tracking lectin-coated beads on the surface and phase-dense features within lamellipodia of MC7 3T3 cells. The movement of these dorsal features occurred at rates approximately three times faster than the rate of movement of the underlying bulk actin cytoskeleton, even when measured in the same individual cells. Thus the transport of these dorsal features must occur by some mechanism other than simple attachment to the moving bulk actin cytoskeleton.

Actin Binding Proteins: Regulation of Cytoskeletal Microfilaments

2003 ◽  
Vol 83 (2) ◽  
pp. 433-473 ◽  
Author(s):  
C. G. Dos Remedios ◽  
D. Chhabra ◽  
M. Kekic ◽  
I. V. Dedova ◽  
M. Tsubakihara ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Binding Proteins ◽  
Complex Structure ◽  
Actin Binding ◽  
Cellular Functions ◽  
Actin Binding Proteins ◽  
Ancestral Gene ◽  
Wide Range ◽  
Human Disorders ◽  
And Function

The actin cytoskeleton is a complex structure that performs a wide range of cellular functions. In 2001, significant advances were made to our understanding of the structure and function of actin monomers. Many of these are likely to help us understand and distinguish between the structural models of actin microfilaments. In particular, 1) the structure of actin was resolved from crystals in the absence of cocrystallized actin binding proteins (ABPs), 2) the prokaryotic ancestral gene of actin was crystallized and its function as a bacterial cytoskeleton was revealed, and 3) the structure of the Arp2/3 complex was described for the first time. In this review we selected several ABPs (ADF/cofilin, profilin, gelsolin, thymosin β4, DNase I, CapZ, tropomodulin, and Arp2/3) that regulate actin-driven assembly, i.e., movement that is independent of motor proteins. They were chosen because 1) they represent a family of related proteins, 2) they are widely distributed in nature, 3) an atomic structure (or at least a plausible model) is available for each of them, and 4) each is expressed in significant quantities in cells. These ABPs perform the following cellular functions: 1) they maintain the population of unassembled but assembly-ready actin monomers (profilin), 2) they regulate the state of polymerization of filaments (ADF/cofilin, profilin), 3) they bind to and block the growing ends of actin filaments (gelsolin), 4) they nucleate actin assembly (gelsolin, Arp2/3, cofilin), 5) they sever actin filaments (gelsolin, ADF/cofilin), 6) they bind to the sides of actin filaments (gelsolin, Arp2/3), and 7) they cross-link actin filaments (Arp2/3). Some of these ABPs are essential, whereas others may form regulatory ternary complexes. Some play crucial roles in human disorders, and for all of them, there are good reasons why investigations into their structures and functions should continue.

scholarly journals Analysis of myofibrillar structure and assembly using fluorescently labeled contractile proteins.

1984 ◽  
Vol 98 (3) ◽  
pp. 825-833 ◽  
Author(s):  
J W Sanger ◽  
B Mittal ◽  
J M Sanger
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
Contractile Proteins ◽  
Actin Binding ◽  
Thin Filaments ◽  
In Vitro System ◽  
Self Association ◽  
Cross Bridges

To study how contractile proteins become organized into sarcomeric units in striated muscle, we have exposed glycerinated myofibrils to fluorescently labeled actin, alpha-actinin, and tropomyosin. In this in vitro system, alpha-actinin bound to the Z-bands and the binding could not be saturated by prior addition of excess unlabeled alpha-actinin. Conditions known to prevent self-association of alpha-actinin, however, blocked the binding of fluorescently labeled alpha-actinin to Z-bands. When tropomyosin was removed from the myofibrils, alpha-actinin then added to the thin filaments as well as the Z-bands. Actin bound in a doublet pattern to the regions of the myosin filaments where there were free cross-bridges i.e., in that part of the A-band free of interdigitating native thin filaments but not in the center of the A-band which lacks cross-bridges. In the presence of 0.1-0.2 mM ATP, no actin binding occurred. When unlabeled alpha-actinin was added first to myofibrils and then labeled actin was added fluorescence occurred not in a doublet pattern but along the entire length of the myofibril. Tropomyosin did not bind to myofibrils unless the existing tropomyosin was first removed, in which case it added to the thin filaments in the l-band. Tropomyosin did bind, however, to the exogenously added tropomyosin-free actin that localizes as a doublet in the A-band. These results indicate that the alpha-actinin present in Z-bands of myofibrils is fully complexed with actin, but can bind exogenous alpha-actinin and, if actin is added subsequently, the exogenous alpha-actinin in the Z-band will bind the newly formed fluorescent actin filaments. Myofibrillar actin filaments did not increase in length when G-actin was present under polymerizing conditions, nor did they bind any added tropomyosin. These observations are discussed in terms of the structure and in vivo assembly of myofibrils.

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