scholarly journals The Ca(2+)-binding domains in non-muscle type alpha-actinin: biochemical and genetic analysis.

1993 ◽  
Vol 121 (3) ◽  
pp. 599-606 ◽  
Author(s):  
W Witke ◽  
A Hofmann ◽  
B Köppel ◽  
M Schleicher ◽  
A A Noegel
Keyword(s):  
Biochemical Data ◽  
Actin Binding ◽  
Regulatory Function ◽  
Cross Linking ◽  
Abnormal Phenotype ◽  
Ef Hand ◽  
Ef Hands ◽  
Overlay Assay

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF-hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
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.

scholarly journals Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

2001 ◽  
Vol 12 (11) ◽  
pp. 3515-3526 ◽  
Author(s):  
Kentaro Nakano ◽  
Kazuomi Satoh ◽  
Akeshi Morimatsu ◽  
Masaaki Ohnuma ◽  
Issei Mabuchi
Keyword(s):  
Actin Cytoskeleton ◽  
Fission Yeast ◽  
Actin Binding ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Depolymerizing Factor ◽  
Binding Domains ◽  
Ef Hand ◽  
Actin Cables

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein.fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

scholarly journals Villin Severing Activity Enhances Actin-based Motility In Vivo

2007 ◽  
Vol 18 (3) ◽  
pp. 827-838 ◽  
Author(s):  
Céline Revenu ◽  
Matthieu Courtois ◽  
Alphée Michelot ◽  
Cécile Sykes ◽  
Daniel Louvard ◽  
...  
Keyword(s):  
Shigella Flexneri ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Loss Of Function ◽  
Mesenchymal Transition ◽  
Capping Protein ◽  
Function Strategy ◽  
In Cells

Villin, an actin-binding protein associated with the actin bundles that support microvilli, bundles, caps, nucleates, and severs actin in a calcium-dependant manner in vitro. We hypothesized that the severing activity of villin is responsible for its reported role in enhancing cell plasticity and motility. To test this hypothesis, we chose a loss of function strategy and introduced mutations in villin based on sequence comparison with CapG. By pyrene-actin assays, we demonstrate that this mutant has a strongly reduced severing activity, whereas nucleation and capping remain unaffected. The bundling activity and the morphogenic effects of villin in cells are also preserved in this mutant. We thus succeeded in dissociating the severing from the three other activities of villin. The contribution of villin severing to actin dynamics is analyzed in vivo through the actin-based movement of the intracellular bacteria Shigella flexneri in cells expressing villin and its severing variant. The severing mutations abolish the gain of velocity induced by villin. To further analyze this effect, we reconstituted an in vitro actin-based bead movement in which the usual capping protein is replaced by either the wild type or the severing mutant of villin. Confirming the in vivo results, villin-severing activity enhances the velocity of beads by more than two-fold and reduces the density of actin in the comets. We propose a model in which, by severing actin filaments and capping their barbed ends, villin increases the concentration of actin monomers available for polymerization, a mechanism that might be paralleled in vivo when an enterocyte undergoes an epithelio-mesenchymal transition.

scholarly journals Monoclonal antibodies specific to a Ca2+-bound form of lipocortin I distinguish its Ca2+-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2

1990 ◽  
Vol 269 (3) ◽  
pp. 709-715 ◽  
Author(s):  
H Hayashi ◽  
M K Owada ◽  
S Sonobe ◽  
K Domae ◽  
T Yamanouchi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Biological Effects ◽  
Free Form ◽  
E Coli ◽  
Phospholipid Binding ◽  
Ef Hand

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

scholarly journals Additional evidence for a proform to tropoelastin from chick aorta

1979 ◽  
Vol 177 (2) ◽  
pp. 559-567 ◽  
Author(s):  
C S Heng-Khoo ◽  
R B Rucker ◽  
K W Buckingham
Keyword(s):  
Basic Protein ◽  
Cross Linking ◽  
Protein Subunit ◽  
Deficient Diet ◽  
Culture In Vitro ◽  
One Step

Evidence is presented for the presence of precursor to tropoelastin in chick arterial extracts. The precursor is approx. 100 000 daltons in size. It is suggested to be a precursor to tropoelastin (72 000 daltons). This protein may be observed in culture in vitro if appropriate precautions are taken to inhibit proteolysis. Once synthesized, it appears to be converted into tropoelastin within 10–20 min. The protein may also be detected in vivo. When 1-day-old cockerels were fed on a copper-deficient diet (less than 1 p.p.m. to inhibit cross-linking) containing epsilon-aminohexanoic acid (0.2%) to retard proteolysis and then injected wiht [3H]valine, extraction of arterial proteins 12h after injection resulted in detection of two major peaks of [3H]valine-labelled protein with pI values of pH 7.0 and 5.0 respectively. The protein that focused at pH 7.0 was estimated to be about 100 000 daltons in size and could be shown to be converted into a more basic protein with the properties of tropoelastin. It is speculated that the protein with pI 5.0 may be yet another extension peptide. The data appear to be in keeping with similar observations by ourselves and others that a proform of tropoelastin exists, and, in at least one step before conversion into tropoelastin, exists as a 100 000-dalton protein subunit.

scholarly journals Ezrin is an Actin Binding Protein That Regulates Sertoli Cell and Spermatid Adhesion During Spermatogenesis

Endocrinology ◽  
2014 ◽  
Vol 155 (10) ◽  
pp. 3981-3995 ◽  
Author(s):  
N. Ece Gungor-Ordueri ◽  
Elizabeth I. Tang ◽  
Ciler Celik-Ozenci ◽  
C. Yan Cheng
Keyword(s):  
Sertoli Cell ◽  
Sertoli Cells ◽  
Adherens Junction ◽  
Actin Binding ◽  
Permeability Barrier ◽  
Tight Junction Permeability ◽  
Residual Bodies ◽  
Cell Interface

Abstract During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.

scholarly journals Antigenicity of the infected-erythrocyte and merozoite surfaces in Falciparum malaria.

1979 ◽  
Vol 150 (5) ◽  
pp. 1241-1254 ◽  
Author(s):  
S G Langreth ◽  
R T Reese
Keyword(s):  
Plasmodium Falciparum ◽  
Falciparum Malaria ◽  
Infected Erythrocyte ◽  
Falciparum Infection ◽  
Human Erythrocytes ◽  
Cross Linking ◽  
Common Antigens

The antigenicity of altered structures induced by Plasmodium falciparum in the membranes of infected Aotus monkey and human erythrocytes was examined. Antisera were obtained from monkeys made immune to malaria. Bound antibodies were shown to be localized on the knob protrusions of infected erythrocytes of both human and monkey origin and from both in vitro and in vivo infections. Therefore, P. falciparum infection has produced similar antigenic changes in the erythrocyte surfaces of both man and monkey. Uninfected erythrocytes and all knobless-infected erythrocytes bound no antibody from immune sera. Strains of P. falciparum from widely different geographic areas that were cultured in vitro in human erythrocytes induced structures (knobs) which have common antigenicity. Merozoites were agglutinated by cross-linking of their cell coats when incubated with immune sera. The binding of ferritin-labeled antibody was heavy on the coats of both homologous and heterologous strains of the parasite, indicating that the merozoite surfaces of these strains share common antigens.

scholarly journals Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Christopher McDonald ◽  
Goran Jovanovic ◽  
Oscar Ces ◽  
Martin Buck
Keyword(s):  
Elastic Stress ◽  
Membrane Binding ◽  
Membrane Integrity ◽  
Regulatory Function ◽  
Membrane Stress ◽  
Inner Membrane ◽  
Thylakoid Biogenesis ◽  
Stress Dependent

ABSTRACTPhage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled,in vitromethodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins’ differing rolesin vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. Thisin vitrorecapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mountedin vivowhen a cell's inner membrane experiences increased SCE stress.IMPORTANCEAll cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response.

scholarly journals The Ubiquitously Expressed Csk Adaptor Protein Cbp Is Dispensable for Embryogenesis and T-Cell Development and Function

2005 ◽  
Vol 25 (23) ◽  
pp. 10533-10542 ◽  
Author(s):  
Marc-Werner Dobenecker ◽  
Christian Schmedt ◽  
Masato Okada ◽  
Alexander Tarakhovsky
Keyword(s):  
T Cell ◽  
Adaptor Protein ◽  
Regulatory Function ◽  
Loss Of Function ◽  
Thymic Development ◽  
Cell Functions ◽  
Carboxy Terminal ◽  
And Function

ABSTRACT Regulation of Src family kinase (SFK) activity is indispensable for a functional immune system and embryogenesis. The activity of SFKs is inhibited by the presence of the carboxy-terminal Src kinase (Csk) at the cell membrane. Thus, recruitment of cytosolic Csk to the membrane-associated SFKs is crucial for its regulatory function. Previous studies utilizing in vitro and transgenic models suggested that the Csk-binding protein (Cbp), also known as phosphoprotein associated with glycosphingolipid microdomains (PAG), is the membrane adaptor for Csk. However, loss-of-function genetic evidence to support this notion was lacking. Herein, we demonstrate that the targeted disruption of the cbp gene in mice has no effect on embryogenesis, thymic development, or T-cell functions in vivo. Moreover, recruitment of Csk to the specialized membrane compartment of “lipid rafts” is not impaired by Cbp deficiency. Our results indicate that Cbp is dispensable for the recruitment of Csk to the membrane and that another Csk adaptor, yet to be discovered, compensates for the loss of Cbp.

Sign in / Sign up

Export Citation Format

Share Document