Cytoskeletal Interactions at the Nuclear Envelope Mediated by Nesprins

Nesprin-1 is a giant tail-anchored nuclear envelope protein composed of an N-terminal F-actin binding domain, a long linker region formed by multiple spectrin repeats and a C-terminal transmembrane domain. Based on this structure, it connects the nucleus to the actin cytoskeleton. Earlier reports had shown that Nesprin-1 binds to nuclear envelope proteins emerin and lamin through C-terminal spectrin repeats. These repeats can also self-associate. We focus on the N-terminal Nesprin-1 sequences and show that they interact with Nesprin-3, a further member of the Nesprin family, which connects the nucleus to the intermediate filament network. We show that upon ectopic expression of Nesprin-3 in COS7 cells, which are nearly devoid of Nesprin-3 in vitro, vimentin filaments are recruited to the nucleus and provide evidence for an F-actin interaction of Nesprin-3 in vitro. We propose that Nesprins through interactions amongst themselves and amongst the various Nesprins form a network around the nucleus and connect the nucleus to several cytoskeletal networks of the cell.

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NUANCE, a giant protein connecting the nucleus and actin cytoskeleton

Journal of Cell Science ◽

10.1242/jcs.115.15.3207 ◽

2002 ◽

Vol 115 (15) ◽

pp. 3207-3222 ◽

Cited By ~ 25

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.

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Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

Journal of Biomedicine and Biotechnology ◽

10.1155/2011/904547 ◽

2011 ◽

Vol 2011 ◽

pp. 1-18

Author(s):

Richard A. Zuellig ◽

Beat C. Bornhauser ◽

Ralf Amstutz ◽

Bruno Constantin ◽

Marcus C. Schaub

Keyword(s):

Actin Cytoskeleton ◽

Tissue Expression ◽

Protein Product ◽

Binding Domain ◽

Actin Binding ◽

Rat Tissues ◽

Cortical Actin ◽

Actin Binding Domain ◽

Spectrin Repeats

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constantsK1=∼5×106andK2=∼1×105 M-1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues.

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The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein

Blood ◽

10.1182/blood.v92.10.3848.422k44_3848_3856 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3848-3856 ◽

Cited By ~ 3

Author(s):

K. Okuda ◽

A. D’Andrea ◽

R.A. Van Etten ◽

J.D. Griffin

Keyword(s):

Transmembrane Domain ◽

Biological Effects ◽

Sh2 Domain ◽

Binding Domain ◽

Erythropoietin Receptor ◽

Myelogenous Leukemia ◽

Actin Binding ◽

Time To Death ◽

Actin Binding Domain

Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

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The C-Terminus of c-Abl Is Required for Proliferation and Viability Signaling in a c-Abl/Erythropoietin Receptor Fusion Protein

Blood ◽

10.1182/blood.v92.10.3848 ◽

1998 ◽

Vol 92 (10) ◽

pp. 3848-3856 ◽

Cited By ~ 3

Author(s):

K. Okuda ◽

A. D’Andrea ◽

R.A. Van Etten ◽

J.D. Griffin

Keyword(s):

Transmembrane Domain ◽

Biological Effects ◽

Sh2 Domain ◽

Binding Domain ◽

Erythropoietin Receptor ◽

Myelogenous Leukemia ◽

Actin Binding ◽

Time To Death ◽

Actin Binding Domain

Abstract Activated ABL oncogenes cause B-cell leukemias in mice and chronic myelogenous leukemia in humans. However, the mechanism of transformation is complex and not well understood. A method to rapidly and reversibly activate c-ABL was created by fusing the extra-cytoplasmic and transmembrane domain of the erythropoietin (EPO) receptor with c-ABL (EPO R/ABL). When this chimeric receptor was expressed in Ba/F3 cells, the addition of EPO resulted in a dose-dependent activation of c-ABL tyrosine kinase and was strongly antiapoptotic and weakly mitogenic. To evaluate the contributions of various ABL domains to biochemical signaling and biological effects, chimeric receptors were constructed in which the ABL SH3 domain was deleted (▵SH3), the SH2 domain was deleted (▵SH2), the C-terminal actin-binding domain was deleted (▵ABD), or kinase activity was eliminated by a point mutation, K290M (KD). The mutant receptors were stably expressed in Ba/F3 cells and analyzed for signaling defects, proliferation, viability, and EPO-induced leukemia in nude mice. When compared with the ability of the full-length EPO R/ABL receptor to induce proliferation and support viability in vitro, the ▵SH3 mutant was equivalent, the ▵SH2 mutant was moderately impaired, and the ▵ABD and KD mutants were profoundly impaired. None of these cell lines caused leukemia in mice in the absence of pharmacological doses of EPO. However, in mice treated with EPO (10 U/d), death from leukemia occurred rapidly with wild-type and ▵SH3. However, time to death was prolonged by at least twofold for ▵SH2 and greater than threefold for ▵ABD. This inducible model of ABL transformation provides a method to link specific signaling defects with specific biological defects and has shown an important role for the C-terminal actin-binding domain in proliferation and transformation in the context of this receptor/oncogene.

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A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

Molecular Biology of the Cell ◽

10.1091/mbc.e06-05-0457 ◽

2006 ◽

Vol 17 (11) ◽

pp. 4720-4735 ◽

Cited By ~ 61

Author(s):

Alistair N. Hume ◽

Abul K. Tarafder ◽

José S. Ramalho ◽

Elena V. Sviderskaya ◽

Miguel C. Seabra

Keyword(s):

Coiled Coil ◽

Binding Domain ◽

Actin Binding ◽

Binding Studies ◽

Null Cell ◽

Coiled Coil Region ◽

Actin Binding Domain ◽

Transport Assay ◽

Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

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Microtubule Actin Cross-Linking Factor (Macf)

The Journal of Cell Biology ◽

10.1083/jcb.147.6.1275 ◽

1999 ◽

Vol 147 (6) ◽

pp. 1275-1286 ◽

Cited By ~ 150

Author(s):

Conrad L. Leung ◽

Dongming Sun ◽

Min Zheng ◽

David R. Knowles ◽

Ronald K.H. Liem

Keyword(s):

Calcium Binding ◽

Coiled Coil ◽

Specific Protein ◽

Binding Domain ◽

Full Length ◽

Actin Binding ◽

Cross Linking ◽

Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

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A High-affinity Interaction with ADP-Actin Monomers Underlies the Mechanism and In Vivo Function of Srv2/cyclase-associated Protein

Molecular Biology of the Cell ◽

10.1091/mbc.e04-06-0444 ◽

2004 ◽

Vol 15 (11) ◽

pp. 5158-5171 ◽

Cited By ~ 74

Author(s):

Pieta K. Mattila ◽

Omar Quintero-Monzon ◽

Jamie Kugler ◽

James B. Moseley ◽

Steven C. Almo ◽

...

Keyword(s):

Site Directed Mutagenesis ◽

Actin Binding ◽

Actin Monomer ◽

Nucleotide Exchange ◽

Actin Organization ◽

C Terminus ◽

Actin Binding Domain ◽

Affinity Interaction

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 μM) compared with ATP-G-actin (Kd 1.9 μM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved β-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.

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The preprophase band-associated kinesin-14 OsKCH2 is a processive minus-end-directed microtubule motor

10.1101/232652 ◽

2017 ◽

Author(s):

Kuo-Fu Tseng ◽

Pan Wang ◽

Yuh-Ru Julie Lee ◽

Joel Bowen ◽

Allison M. Gicking ◽

...

Keyword(s):

Coiled Coil ◽

Preprophase Band ◽

Cytoplasmic Dynein ◽

Coiled Coils ◽

Land Plants ◽

Actin Binding ◽

Actin Binding Domain ◽

Microtubule Motor

AbstractIn animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.

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Utrophin actin binding domain: analysis of actin binding and cellular targeting

Journal of Cell Science ◽

10.1242/jcs.108.1.63 ◽

1995 ◽

Vol 108 (1) ◽

pp. 63-71 ◽

Cited By ~ 16

Author(s):

S.J. Winder ◽

L. Hemmings ◽

S.K. Maciver ◽

S.J. Bolton ◽

J.M. Tinsley ◽

...

Keyword(s):

Binding Domain ◽

Actin Binding ◽

Muscle Actin ◽

Dystrophic Muscle ◽

Developmentally Regulated ◽

Glycoprotein Complex ◽

Muscle Tissues ◽

Actin Binding Domain ◽

Acid Domain

Utrophin, or dystrophin-related protein, is an autosomal homologue of dystrophin. The protein is apparently ubiquitously expressed and in muscle tissues the expression is developmentally regulated. Since utrophin has a similar domain structure to dystrophin it has been suggested that it could substitute for dystrophin in dystrophic muscle. Like dystrophin, utrophin has been shown to be associated with a membrane-bound glycoprotein complex. Here we demonstrate that expressed regions of the predicted actin binding domain in the NH2 terminus of utrophin are able to bind to F-actin in vitro, but do not interact with G-actin. The utrophin actin binding domain was also able to associate with actin-containing structures, stress fibres and focal contacts, when microinjected into chick embryo fibroblasts. The expressed NH2-terminal 261 amino acid domain of utrophin has an affinity for skeletal F-action (Kd 19 +/- 2.8 microM), midway between that of the corresponding domains of alpha-actinin (Kd 4 microM) and dystrophin (Kd 44 microM). Moreover, this utrophin domain binds to non-muscle actin with a approximately 4-fold higher affinity than to skeletal muscle actin. These data (together with those of Matsumura et al. (1992) Nature, 360, 588–591) demonstrate for the first time that utrophin is capable of performing a functionally equivalent role to that of dystrophin. The NH2 terminus of utrophin binds to actin and the COOH terminus binds to the membrane associated glycoprotein complex, thus in non-muscle and developing muscle utrophin performs the same predicted ‘spacer’ or ‘shock absorber’ role as dystrophin in mature muscle tissues. These data suggest that utrophin could replace dystrophin functionally in dystrophic muscle.

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Physical and genetic interaction of filamin with presenilin in Drosophila

Journal of Cell Science ◽

10.1242/jcs.113.19.3499 ◽

2000 ◽

Vol 113 (19) ◽

pp. 3499-3508 ◽

Cited By ~ 1

Author(s):

Y. Guo ◽

S.X. Zhang ◽

N. Sokol ◽

L. Cooley ◽

G.L. Boulianne

Keyword(s):

Transmembrane Domain ◽

Genetic Interaction ◽

Amino Acid Identity ◽

Binding Domain ◽

Actin Binding ◽

Loop Region ◽

Actin Binding Domain ◽

Alternatively Spliced ◽

The Central Nervous System ◽

Hydrophilic Loop

Presenilins were first identified as causative factors in early onset, familial Alzheimer's Disease (FAD). They are predicted to encode a highly conserved novel family of eight transmembrane domain proteins with a large hydrophilic loop between TM6 and TM7 that is the site of numerous FAD mutations. Here, we show that the loop region of Drosophila and human presenilins interacts with the C-terminal domain of Drosophila filamin. Furthermore, we show that Drosophila has at least two major filamin forms generated by alternative splicing from a gene that maps to position 89E10-89F4 on chromosome 3. The longest form is enriched in the central nervous system and ovaries, shares 41.7% overall amino acid identity with human filamin (ABP-280) and contains an N-terminal actin-binding domain. The shorter form is broadly expressed and encodes an alternatively spliced form of the protein lacking the actin-binding domain. Finally, we show that presenilin and filamin are expressed in overlapping patterns in Drosophila and that dominant adult phenotypes produced by overexpression of presenilin can be suppressed by overexpression of filamin in the same tissue. Taken together, these results suggest that presenilin and filamin functionally interact during development.

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