scholarly journals Golgi Localization of Syne-1

2003 ◽  
Vol 14 (6) ◽  
pp. 2410-2424 ◽  
Author(s):  
Lisa Lucio Gough ◽  
Jun Fan ◽  
Stephen Chu ◽  
Shawn Winnick ◽  
Kenneth A. Beck
Keyword(s):  
Amino Acids ◽  
Nuclear Envelope ◽  
Sequence Data ◽  
Dominant Negative ◽  
Actin Binding ◽  
Expression Library ◽  
Genome Database ◽  
Binding Domains ◽  
Large Gene ◽  
Golgi Localization

We have previously identified a Golgi-localized spectrin isoform by using an antibody to the β-subunit of erythrocyte spectrin. In this study, we show that a screen of a λgt11 expression library resulted in the isolation of an ∼5-kb partial cDNA from a Madin-Darby bovine kidney (MDBK) cell line, which encoded a polypeptide of 1697 amino acids with low, but detectable, sequence homology to spectrin (37%). A blast search revealed that this clone overlaps with the 5′ end of a recently identified spectrin family member Syne-1B/Nesprin-1β, an alternately transcribed gene with muscle-specific forms that bind acetylcholine receptor and associate with the nuclear envelope. By comparing the sequence of the MDBK clone with sequence data from the human genome database, we have determined that this cDNA represents a central portion of a very large gene (∼500 kb), encoding an ∼25-kb transcript that we refer to as Syne-1. Syne-1 encodes a large polypeptide (8406 amino acids) with multiple spectrin repeats and a region at its amino terminus with high homology to the actin binding domains of conventional spectrins. Golgi localization for this spectrin-like protein was demonstrated by expression of epitope-tagged fragments in MDBK and COS cells, identifying two distinct Golgi binding sites, and by immunofluorescence microscopy by using several different antibody preparations. One of the Golgi binding domains on Syne-1 acts as a dominant negative inhibitor that alters the structure of the Golgi complex, which collapses into a condensed structure near the centrosome in transfected epithelial cells. We conclude that the Syne-1 gene is expressed in a variety of forms that are multifunctional and are capable of functioning at both the Golgi and the nuclear envelope, perhaps linking the two organelles during muscle differentiation.

Patterns of evolutionary conservation in the nesprin genes highlight probable functionally important protein domains and isoforms

2008 ◽  
Vol 36 (6) ◽  
pp. 1359-1367 ◽  
Author(s):  
Jennifer G. Simpson ◽  
Roland G. Roberts
Keyword(s):  
Nuclear Envelope ◽  
Expressed Sequence Tag ◽  
Sequence Data ◽  
Actin Binding ◽  
Structural Elements ◽  
Spectrin Repeat ◽  
Protein Protein Interaction ◽  
Common Structure ◽  
Genes Encoding ◽  
Membrane Bound

The nesprins [also known as SYNEs (synaptic nuclear envelope proteins)] are a family of type II transmembrane proteins implicated in the tethering of membrane-bound organelles and in the genetic aetiology of cerebellar ataxia and Emery–Dreifuss muscular dystrophy. They are characterized by a common structure of an SR (spectrin repeat) rod domain and a C-terminal transmembrane KLS (klarsicht)/KASH [klarsicht/ANC-1 (anchorage 1)/SYNE homology] domain which interacts with SUN [Sad1p/UNC (uncoordinated)-84] proteins in the nuclear envelope; most nesprins also have N-terminal actin-binding CH (calponin homology) domains. The genes encoding the three vertebrate nesprins (five in bony fish) and the small transmembrane actin-binding protein calmin are related to each other by ancient duplications and rearrangements. In the present paper, we collate sequence data for nesprins and calmins across the vertebrate clade and use these to study evolutionary constraints acting on their genes. We show that the rod domains of the larger nesprins are composed almost entirely of unbroken SR-like structures (74 in nesprin-1 and 56 in nesprin-2) and that these range from poorly conserved purely structural elements to highly conserved regions with a presumed protein–protein interaction function. The analysis suggests several interesting regions for future study. We also assess the evolutionary and EST (expressed sequence tag) expression support for nesprin isoforms, both known and novel; our findings suggest that substantial reassessment is required.

scholarly journals Interactions of tensin with actin and identification of its three distinct actin-binding domains.

1994 ◽  
Vol 125 (5) ◽  
pp. 1067-1075 ◽  
Author(s):  
S H Lo ◽  
P A Janmey ◽  
J H Hartwig ◽  
L B Chen
Keyword(s):  
Amino Acids ◽  
Actin Filaments ◽  
Actin Polymerization ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Molar Ratio ◽  
Actin Assembly ◽  
Binding Domains ◽  
Focal Contacts ◽  
End Capping

Tensin, a 200-kD phosphoprotein of focal contacts, contains sequence homologies to Src (SH2 domain), and several actin-binding proteins. These features suggest that tensin may link the cell membrane to the cytoskeleton and respond directly to tyrosine kinase signalling pathways. Here we identify three distinct actin-binding domains within tensin. Recombinant tensin purified after overexpression by a baculovirus system binds to actin filaments with Kd = 0.1 microM, cross-links actin filaments at a molar ratio of 1:10 (tensin/actin), and retards actin assembly by barbed end capping with Kd = 20 nM. Tensin fragments were constructed and expressed as fusion proteins to map domains having these activities. Three regions from tensin interact with actin: two regions composed of amino acids 1 to 263 and 263 to 463, cosediment with F-actin but do not alter the kinetics of actin assembly; a region composed of amino acids 888-989, with sequence homology to insertin, retards actin polymerization. A claw-shaped tensin dimer would have six potential actin-binding sites and could embrace the ends of two actin filaments at focal contacts.

scholarly journals Lamin A/C–dependent Localization of Nesprin-2, a Giant Scaffolder at the Nuclear Envelope

2005 ◽  
Vol 16 (7) ◽  
pp. 3411-3424 ◽  
Author(s):  
Thorsten Libotte ◽  
Hafida Zaim ◽  
Sabu Abraham ◽  
V. C. Padmakumar ◽  
Maria Schneider ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Normal Skin ◽  
Basal Layer ◽  
Dominant Negative ◽  
Actin Binding ◽  
Lamin A ◽  
Common Region ◽  
Lamin B

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of α-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.

scholarly journals The Actin-Binding Domain of Slac2-a/Melanophilin Is Required for Melanosome Distribution in Melanocytes

2003 ◽  
Vol 23 (15) ◽  
pp. 5245-5255 ◽  
Author(s):  
Taruho S. Kuroda ◽  
Hiroyoshi Ariga ◽  
Mitsunori Fukuda
Keyword(s):  
Amino Acids ◽  
Binding Domain ◽  
Dominant Negative ◽  
Actin Binding ◽  
Dominant Negative Effect ◽  
C Terminus ◽  
Defective Mutant ◽  
Actin Binding Domain ◽  
Negative Effect ◽  
Myosin Va

ABSTRACT Melanosomes containing melanin pigments are transported from the cell body of melanocytes to the tips of their dendrites by a combination of microtubule- and actin-dependent machinery. Three proteins, Rab27A, myosin Va, and Slac2-a/melanophilin (a linker protein between Rab27A and myosin Va), are known to be essential for proper actin-based melanosome transport in melanocytes. Although Slac2-a directly interacts with Rab27A and myosin Va via its N-terminal region (amino acids 1 to 146) and the middle region (amino acids 241 to 405), respectively, the functional importance of the putative actin-binding domain of the Slac2-a C terminus (amino acids 401 to 590) in melanosome transport has never been elucidated. In this study we showed that formation of a tripartite protein complex between Rab27A, Slac2-a, and myosin Va alone is insufficient for peripheral distribution of melanosomes in melanocytes and that the C-terminal actin-binding domain of Slac2-a is also required for proper melanosome transport. When a Slac2-a deletion mutant (ΔABD) or point mutant (KA) that lacks actin-binding ability was expressed in melanocytes, the Slac2-a mutants induced melanosome accumulation in the perinuclear region, possibly by a dominant negative effect, the same as the Rab27A-binding-defective mutant of Slac2-a or the myosin Va-binding-defective mutant. Our findings indicate that Slac2-a organizes actin-based melanosome transport in cooperation with Rab27A, myosin Va, and actin.

In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1617-1630
Author(s):  
Suk-Won Jin ◽  
Nancy Arno ◽  
Adam Cohen ◽  
Amy Shah ◽  
Qijin Xu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Germ Cell ◽  
Rna Binding ◽  
Dominant Negative ◽  
Missense Mutations ◽  
Biochemical Tests ◽  
Cell Fates ◽  
Cytoplasmic Polyadenylation ◽  
Binding Domains ◽  
Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

scholarly journals Isolation and properties of two actin-binding domains in gelsolin.

1985 ◽  
Vol 260 (28) ◽  
pp. 15232-15238 ◽  
Author(s):  
D J Kwiatkowski ◽  
P A Janmey ◽  
J E Mole ◽  
H L Yin
Keyword(s):  
Actin Binding ◽  
Binding Domains

Osmotic stress-induced remodeling of the cortical cytoskeleton

2002 ◽  
Vol 283 (3) ◽  
pp. C850-C865 ◽  
Author(s):  
Caterina Di Ciano ◽  
Zilin Nie ◽  
Katalin Szászi ◽  
Alison Lewis ◽  
Takehito Uruno ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
De Novo ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Actin Binding ◽  
Cell Cortex ◽  
Actin Assembly ◽  
Signaling Mechanism ◽  
Actin Binding Domain ◽  
Cytoskeleton Remodeling

Osmotic stress is known to affect the cytoskeleton; however, this adaptive response has remained poorly characterized, and the underlying signaling pathways are unexplored. Here we show that hypertonicity induces submembranous de novo F-actin assembly concomitant with the peripheral translocation and colocalization of cortactin and the actin-related protein 2/3 (Arp2/3) complex, which are key components of the actin nucleation machinery. Additionally, hyperosmolarity promotes the association of cortactin with Arp2/3 as revealed by coimmunoprecipitation. Using various truncation or phosphorylation-incompetent mutants, we show that cortactin translocation requires the Arp2/3- or the F-actin binding domain, but the process is independent of the shrinkage-induced tyrosine phosphorylation of cortactin. Looking for an alternative signaling mechanism, we found that hypertonicity stimulates Rac and Cdc42. This appears to be a key event in the osmotically triggered cytoskeletal reorganization, because 1) constitutively active small GTPases translocate cortactin, 2) Rac and cortactin colocalize at the periphery of hypertonically challenged cells, and 3) dominant-negative Rac and Cdc42 inhibit the hypertonicity-provoked cortactin and Arp3 translocation. The Rho family-dependent cytoskeleton remodeling may be an important osmoprotective response that reinforces the cell cortex.

scholarly journals Mammalian microtubule P-body dynamics are mediated by nesprin-1

2014 ◽  
Vol 205 (4) ◽  
pp. 457-475 ◽  
Author(s):  
Dipen Rajgor ◽  
Jason A. Mellad ◽  
Daniel Soong ◽  
Jerome B. Rattner ◽  
Marvin J. Fritzler ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Nuclear Envelope ◽  
Sodium Arsenite ◽  
Sr Proteins ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Processing Bodies ◽  
Spectrin Repeat ◽  
Microtubule Attachment ◽  
Body Dynamics

Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50Nesp1, a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50Nesp1 caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB–microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50Nesp1 was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50Nesp1 as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability

1987 ◽  
Vol 7 (8) ◽  
pp. 2783-2793
Author(s):  
S J Elledge ◽  
R W Davis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Dna Damage ◽  
Ribonucleotide Reductase ◽  
Small Subunit ◽  
Cross Reaction ◽  
Expression Library ◽  
Gene Encoding ◽  
Ribonucleotide Reductases ◽  
Colony Color

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.

The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽  
1999 ◽  
Vol 126 (13) ◽  
pp. 2841-2853 ◽  
Author(s):  
G. Deshpande ◽  
G. Calhoun ◽  
P.D. Schedl
Keyword(s):  
Fusion Protein ◽  
Rna Binding ◽  
Chimeric Protein ◽  
Dominant Negative ◽  
Terminal Domain ◽  
Binding Domains ◽  
Transcriptional Regulatory ◽  
Dominant Negative Activity ◽  
Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

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