scholarly journals Polarity of flagellar assembly in Chlamydomonas.

1992 ◽  
Vol 119 (6) ◽  
pp. 1605-1611 ◽  
Author(s):  
K A Johnson ◽  
J L Rosenbaum
Keyword(s):  
Cell Fusion ◽  
Full Length ◽  
Wild Type ◽  
Alpha Tubulin ◽  
Radial Spoke ◽  
Donor Cells ◽  
Radial Spokes ◽  
Flagellar Assembly ◽  
Series Of Experiments

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.

scholarly journals Radial spokes of Chlamydomonas flagella: polypeptide composition and phosphorylation of stalk components.

1981 ◽  
Vol 88 (1) ◽  
pp. 73-79 ◽  
Author(s):  
G Piperno ◽  
B Huang ◽  
Z Ramanis ◽  
D J Luck
Keyword(s):  
Cellular Proteins ◽  
Two Dimensional ◽  
Wild Type ◽  
Molecular Weight Range ◽  
Radial Spoke ◽  
Radial Spokes ◽  
32P Incorporation ◽  
Electrophoretic Techniques ◽  
Dynein Arms

Polypeptides from flagella or axonemes of Chlamydomonas reinhardtii were analyzed by labeling cellular proteins by prolonged growth on 35S-containing media and using one- and two-dimensional electrophoretic techniques which can resolve greater than 170 axonemal components. By this approach, a paralyzed mutant that lacks axonemal radial spokes, pf14, has been shown to lack 17 polypeptides in the molecular weight range of 20,000 to 124,000 and in the isoelectric point range of 4.8-7.1. Five of those polypeptides are also missing in the mutant pf-1 which lacks only radial spokeheads. The identification of the 17 polypeptides missing in pf-14 as components of radial spoke structures and the localization of the polypeptides lacking in pf-1 within the spokehead, are supported by experiments of chemical dissection of wild-type axonemes. Extraction procedures that solubilize outer and inner dynein arms preserve the structure of the radial spokes along with the 17 polypeptides in question. Six radial spoke polypeptides are solubilized in conditions that cause disassembly of radial spokeheads from the stalks and those components include the five polypeptides missing in pf-1. No Ca++- or Mg++-activated ATPase activities were found to be associated with solubilized preparations of wild-type radial spokeheads. In vivo pulse 32P incorporation experiments provide evidence that greater than 80 axonemal components are labeled by 32P and that five of the radial spoke stalk polypeptides are modified to different extents.

scholarly journals A conserved CaM- and radial spoke–associated complex mediates regulation of flagellar dynein activity

2007 ◽  
Vol 179 (3) ◽  
pp. 515-526 ◽  
Author(s):  
Erin E. Dymek ◽  
Elizabeth F. Smith
Keyword(s):  
Mass Spectrometry ◽  
Sequence Similarity ◽  
Second Messengers ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Calcium Sensor ◽  
Wild Type ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Dynein Arms

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.

scholarly journals Rab27a Is an Essential Component of Melanosome Receptor for Myosin Va

2002 ◽  
Vol 13 (5) ◽  
pp. 1735-1749 ◽  
Author(s):  
Xufeng Wu ◽  
Fei Wang ◽  
Kang Rao ◽  
James R. Sellers ◽  
John A. Hammer
Keyword(s):  
Full Length ◽  
Tail Domain ◽  
Wild Type ◽  
Essential Component ◽  
Myosin Va ◽  
Alternatively Spliced ◽  
Additional Protein ◽  
The Brain ◽  
Alternatively Spliced Exons

Melanocytes that lack the GTPase Rab27a (ashen) are disabled in myosin Va-dependent melanosome capture because the association of the myosin with the melanosome surface depends on the presence of this resident melanosomal membrane protein. One interpretation of these observations is that Rab27a functions wholly or in part as the melanosome receptor for myosin Va (Myo5a). Herein, we show that the ability of the myosin Va tail domain to localize to the melanosome and generate a myosin Va null (dilute) phenotype in wild-type melanocytes is absolutely dependent on the presence of exon F, one of two alternatively spliced exons present in the tail of the melanocyte-spliced isoform of myosin Va but not the brain-spliced isoform. Exon D, the other melanocyte-specific tail exon, is not required. Similarly, the ability of full-length myosin Va to colocalize with melanosomes and to rescue their distribution indilute melanocytes requires exon F but not exon D. These results predict that an interaction between myosin Va and Rab27a should be exon F dependent. Consistent with this, Rab27a present in detergent lysates of melanocytes binds to beads coated with purified, full-length melanocyte myosin Va and melanocyte myosin Va lacking exon D, but not to beads coated with melanocyte myosin Va lacking exon F or brain myosin Va. Moreover, the preparation of melanocyte lysates in the presence of GDP rather than guanosine-5′-O-(3-thio)triphosphate reduces the amount of Rab27a bound to melanocyte myosin Va-coated beads by approximately fourfold. Finally, pure Rab27a does not bind to myosin Va-coated beads, suggesting that these two proteins interact indirectly. Together, these results argue that Rab27a is an essential component of a protein complex that serves as the melanosome receptor for myosin Va, suggest that this complex contains at least one additional protein capable of bridging the indirect interaction between Rab27a and myosin Va, and imply that the recruitment of myosin Va to the melanosome surface in vivo should be regulated by factors controlling the nucleotide state of Rab27a.

scholarly journals Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function.

1981 ◽  
Vol 88 (1) ◽  
pp. 80-88 ◽  
Author(s):  
B Huang ◽  
G Piperno ◽  
Z Ramanis ◽  
D J Luck
Keyword(s):  
Gene Product ◽  
Biochemical Analysis ◽  
Indirect Evidence ◽  
Mutant Gene ◽  
Molecular Defect ◽  
Molecular Weight Range ◽  
Radial Spoke ◽  
Radial Spokes ◽  
And Function

In addition to the previously studied pf-14 and pf-1 loci in Chlamydomonas reinhardtii, mutations for another five genes (pf-17, pf-24, pf-25, pf-26, and pf-27) have been identified and characterized as specifically affecting the assembly and function of the flagellar radial spokes. Mutants for each of the newly identified loci show selective alterations for one or more of the 17 polypeptides in the molecular weight range of 20,000-130,000 which form the radial spoke structure. In specific instances the molecular defect has been correlated with altered radial spoke morphology. Biochemical analysis of in vivo complementation in mutant X wild-type dikaryons has provided indirect evidence that mutations for four of the five new loci (pf-17, pf-24, pf-25, and pf-26) reside in structural genes for spoke components. In the case of pf-24, the identity of the mutant gene product was supported by analysis of induced intragenic revertants. In contrast to the other radial spoke mutants thus far investigated, evidence suggests that the gene product in pf-27 is extrinsic to the radial spokes and is required for the specific in vivo phosphorylation of spoke polypeptides.

Sto1p, a fission yeast protein similar to tubulin folding cofactor E, plays an essential role in mitotic microtubule assembly

1999 ◽  
Vol 112 (12) ◽  
pp. 1979-1988 ◽  
Author(s):  
E.L. Grishchuk ◽  
J.R. McIntosh
Keyword(s):  
Fission Yeast ◽  
Microtubule Assembly ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Cold Sensitive ◽  
Alpha Beta ◽  
Sensitive Allele ◽  
Delta Cells

The proper functioning of microtubules depends crucially on the availability of polymerizable alpha/beta tubulin dimers. Their production occurs concomitant with the folding of the tubulin polypeptides and is accomplished in part by proteins known as Cofactors A through E. In the fission yeast, Schizosaccharomyces pombe, this tubulin folding pathway is essential. We have taken advantage of the excellent cytology available in S. pombe to examine the phenotypic consequences of a deletion of sto1(+), a gene that encodes a protein similar to Cofactor E, which is required for the folding of alpha-tubulin. The interphase microtubule cytoskeleton in sto1-delta cells is severely disrupted, and as cells enter mitosis their spindles fail to form. After a transient arrest with condensed chromosomes, the cells exit mitosis and resume DNA synthesis, whereupon they septate abnormally and die. Overexpression of Spo1p is toxic to cells carrying a cold-sensitive allele of the alpha- but not the beta-tubulin gene, consistent with the suggestion that this protein plays a role like that of Cofactor E. Unlike its presumptive partner Cofactor D (Alp1p), however, Sto1p does not localize to microtubules but is found throughout the cell. Overexpression of Sto1p has no toxic effects in wild-type cells, suggesting that it is unable to disrupt alpha/beta tubulin dimers in vivo.

Selective disruption of E-cadherin function in early Xenopus embryos by a dominant negative mutant

Development ◽  
1994 ◽  
Vol 120 (4) ◽  
pp. 901-909 ◽  
Author(s):  
E. Levine ◽  
C.H. Lee ◽  
C. Kintner ◽  
B.M. Gumbiner
Keyword(s):  
Early Embryo ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Truncated Form ◽  
Adhesive Properties ◽  
Negative Mutant ◽  
E Cadherin

E-cadherin function was disrupted in vivo in developing Xenopus laevis embryos through the expression of a mutant E-cadherin protein lacking its cytoplasmic tail. This truncated form of E-cadherin was designed to act as a dominant negative mutant by competing with the extracellular interactions of wild-type endogenous E-cadherin. Expression of truncated E-cadherin in the early embryo causes lesions to develop in the ectoderm during gastrulation. In contrast, expression of a similarly truncated N-cadherin protein failed to cause the lesions. The ectodermal defect caused by the truncated E-cadherin is rescued by overexpression of wild-type E-cadherin, by co-injection of full-length E-cadherin RNA along with the RNA for the truncated form. Overexpression of full-length C-cadherin, however, is unable to compensate for the disruption of E-cadherin function and can actually cause similar ectodermal lesions when injected alone, suggesting that there is a specific requirement for E-cadherin. Therefore, E-cadherin seems to be specifically required for maintaining the integrity of the ectoderm during epiboly in the gastrulating Xenopus embryo. Differential cadherin expression reflects, therefore, the requirement for distinct adhesive properties during different morphogenetic cell behaviors.

Low VWF:RCo In Subjects with VWF Polymorphisms D1472H and P1467S Due to Decreased Binding of Ristocetin to the VWF A1 Domain

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2208-2208
Author(s):  
Veronica H Flood ◽  
Jeffrey S Wren ◽  
Sandra L Haberichter ◽  
Kenneth D Friedman ◽  
Daniel B. Bellissimo ◽  
...  
Keyword(s):  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Specific Interaction ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Wild Type ◽  
Microtiter Plates ◽  
A1 Domain ◽  
Von Willebrand

Abstract Abstract 2208 Diagnosis of von Willebrand disease (VWD) currently relies on two assays of von Willebrand factor (VWF), the VWF antigen ELISA (VWF:Ag) and the VWF ristocetin cofactor activity assay (VWF:RCo). The latter exploits the capacity of ristocetin to induce VWF – platelet interactions as a measure of VWF function. Ristocetin, however, is a non-physiologic agonist as shear stress is the physiologic stimulus inducing VWF to bind platelets in vivo. Recently we have reported that the VWF:RCo/VWF:Ag ratio is decreased in individuals with an A1 domain polymorphism, D1472H. The lack of bleeding in subjects with this polymorphism suggests D1472H does not create a physiologic defect in VWF – platelet interactions. D1472H is directly adjacent to a known ristocetin-binding area in the VWF A1 region (Leu 1457 – Pro 1471), supporting the hypothesis that D1472H affects the ability of ristocetin to bind VWF. Similarly, a heterozygous sequence change leading to P1467S (located in the same ristocetin binding domain) resulted in an undetectable VWF:RCo but no bleeding symptoms were noted in affected subjects. To further investigate the cause of this observation, we developed a method to study the binding of ristocetin to VWF directly. Maleic anhydride microtiter plates were used to capture ristocetin via its amine groups. A VWF source, either plasma or recombinant VWF (rVWF), was then added, wells washed, and VWF binding detected using anti-VWF antibodies. Both plasma and rVWF bound to the captured ristocetin similarly with ristocetin plating concentrations ranging from 0.01 to 1 mg/ml. Specificity of ristocetin dependent VWF binding was confirmed, as preincubation of ristocetin with rVWF decreased binding of rVWF to immobilized solid-phase ristocetin. No detectable binding was present for a full length rVWF construct with the entire A1 loop deleted (del 1242–1478) or a construct missing part of the A1 loop (del 1392–1402). VWF binding to ristocetin was inhibited by both monoclonal and polyclonal antibodies directed against the VWF A1 loop. VWF A1 loop constructs with the A1 domain polymorphisms D1472H and P1467S showed decreased binding to ristocetin when compared to a wild-type A1 loop construct. Wild-type A1 loop binding to ristocetin in our assay was 0.98 while 1472H A1 loop binding was reduced at 0.77 (p<0.001 compared to wild-type) and 1467S A1 loop binding was 0.45 (p<0.001 compared to wild-type). No difference was seen when full length VWF constructs with these polymorphisms were studied, perhaps due to other ristocetin recognition sequences present in VWF as reported by Scott and colleagues (JBC 1991). Binding of VWF to vancomycin, a glycopeptide antibiotic similar to ristocetin that does not induce platelet agglutination, was normal. The ristocetin-specific interaction with VWF may require a specific structural conformation of the A1 loop. These results suggest that VWF A1 domain polymorphisms, or the disruption of the entire A1 loop, have the ability to interfere with VWF – ristocetin interactions independent from VWF – platelet interactions. Clinical assays of VWF function that depend on ristocetin may therefore provide misleading results for subjects with VWF polymorphisms affecting ristocetin binding. Disclosures: Montgomery: GTI Diagnostics, Inc: Consultancy.

Role Of Connexin 32 In Hematopoiesis: Maintaining Quiescence Of Hematopoietic Stem Cells and Their Proliferation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1185-1185
Author(s):  
Yoko Hirabayashi ◽  
Isao Tsuboi ◽  
Byung-Il Yoon ◽  
Jun Kanno ◽  
James Trosko ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Average Percentage ◽  
Type Group ◽  
Donor Cells ◽  
In The Wild

Abstract Previously, we reported the findings of our studies on the role of connexin (Cx) 32 during steady-state hematopoiesis and its potential protective role against leukemogenesis. Namely, in wild-type mice, Cx32 expression was solely detected in primitive hematopoietic stem/progenitor cells (HSCs/HPCs). Since Cxs are essential molecules for multicellular organisms, Cxs are surmised to be present in the hematopoietic tissue to facilitate cell-cell communication between HSCs/HPCs themselves rather than between HSCs/HPCs and stromal cells. In addition, Cx32-knockout (KO) mice showed the following characteristics: first, a prominent decrease in the number of peripheral mononuclear cells (PMCs) associated with various HPCs; second, a significant increase in the number of HSCs, at least until 20 weeks of age; and third, an apparently delayed regeneration of HPCs after chemical abrasion. Furthermore, the incidence of leukemogenicity induced by methylnitrosourea increased prominently. These possible leukemogenic propensities taken together imply that Cx32 plays an important role in maintaining steady-state hematopoiesis and in suppressing leukemogenesis. In this study, first, we examined the cell kinetics of HPCs [CFU-GM, colony forming unit (CFU) granulomacrophage; CFU-S9/CFU-S13, CFU in spleen on day 9/13] by evaluating the percentage of cycling HPCs with continuous incorporation of bromodeoxyuridine (BrdUrd) in vivo for up to 3 months, followed by exposure to ultraviolet-A to eliminate cells that incorporated BrdUrd. Using this method with the continuous incorporation of BrdUrd in vivo in mice up to 1.5 years of age, we discovered the existence of a long-term and stable, dormant fraction in the HPCs of wild-type mice. Without Cx32, the percentage of the entire cycling fraction of CFU-S13 apparently increased continuously, which indicates that Cx32 could restore the quiescence of hematopoiesis and thereby maintain HSCs/HPCs. This is consistent with the findings that the number of HPCs increased and the number of HSCs decreased with aging in Cx32-KO mice. Next, we examined the bone-marrow reconstitution capability of HSCs of Cx32-KO mice by serial transplantation. Five hundred cells in the lineage-negative, c-kit-positive, and Sca1-positive (LKS) fraction isolated from wild-type and Cx32-KO mice (Ly5.2) were transplanted into lethally irradiated first recipients (Ly5.1) separately with 2x105 freshly isolated bone marrow cells from wild-type F1(Ly5.1/Ly5.2) mice as rescuing cells, which prevent acute radiation injury. Two months after transplantation, both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters, although mice reconstituted with cells in the LKS fraction without Cx32 showed a rather higher average percentage of donor-origin PMCs and a lower average percentage of the donor-origin cells in the LKS fraction than those with Cx32 (wild-type group vs. Cx32-KO group, average percentage of Ly5.2 with respect to the total ± standard deviations (s.d.); PMCs, 34.7±14.3% vs. 50.7±9.9 %, p=0.006; cells in the LKS fraction, 20.8±5.2% vs. 14.2±5.3%, p=0.110). Then, donor cells (Ly5.2) in the LKS fraction were isolated from the primary recipients of both groups separately and 400 cells were transferred into each secondary recipient (Ly5.1) with 2x105 rescuing cells from F1(Ly5.1/Ly5.2) mice. Although secondary recipients from both groups showed reconstituted hematopoiesis without any significant differences in various hematopoietic parameters four months after reconstitution, similar to the primary recipients, cells of donor origin in the LKS fraction could be detected only in the wild-type group. Namely, four out of seven recipients in the wild-type group showed over 0.5% donor cells and the average percentage and s.d. for 4 mice was 25.1±27.9%, whereas none of the recipients out of five in the Cx32-KO group showed more than 0.5%. The above-mentioned findings in this study in addition to the previous findings imply that Cx32 plays an essential role in maintaining self-renewal proliferation of primitive hematopoietic stem cells to prevent their exhaustion, and also in suppressing neoplastic changes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Role for CCR5 in Dissemination of Vaccinia Virus In Vivo

2008 ◽  
Vol 83 (5) ◽  
pp. 2226-2236 ◽  
Author(s):  
Ramtin Rahbar ◽  
Thomas T. Murooka ◽  
Eleanor N. Fish
Keyword(s):  
T Cells ◽  
Vaccinia Virus ◽  
Time Course ◽  
Lymphatic System ◽  
Wild Type ◽  
Intranasal Inoculation ◽  
Viral Loads ◽  
Human T Cells ◽  
Series Of Experiments

ABSTRACT In an earlier report, we provided evidence that expression of CCR5 by primary human T cells renders them permissive for vaccinia virus (VACV) replication. This may represent a mechanism for dissemination throughout the lymphatic system. To test this hypothesis, wild-type CCR5+/+ and CCR5 null mice were challenged with VACV by intranasal inoculation. In time course studies using different infective doses of VACV, we identified viral replication in the lungs of both CCR5+/+ and CCR5−/− mice, yet there were diminished viral loads in the spleens and brains of CCR5−/− mice compared with CCR5+/+ mice. Moreover, in association with VACV infection, we provide evidence for CD4+ and CD8+ T-cell as well as CD11c+ and F4/80+ cell infiltration into the lungs of CCR5+/+ but not CCR5−/− mice, and we show that the CCR5-expressing T cells harbor virus. We demonstrate that this CCR5 dependence is VACV specific, since CCR5−/− mice are as susceptible to intranasal influenza virus (A/WSN/33) infection as CCR5+/+ mice. In a final series of experiments, we provide evidence that adoptive transfer of CCR5+/+ bone marrow leukocytes into CCR5−/− mice restores VACV permissiveness, with evidence of lung and spleen infection. Taken together, our data suggest a novel role for CCR5 in VACV dissemination in vivo.

scholarly journals Use of interleukin 7 receptor-α knockout donor cells demonstrates the lymphoid independence of dendritic cells

Blood ◽  
2006 ◽  
Vol 107 (1) ◽  
pp. 184-186 ◽  
Author(s):  
Satoshi Takeuchi ◽  
Stephen I. Katz
Keyword(s):  
Dendritic Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Langerhans Cells ◽  
Wild Type ◽  
Lymphoid Lineage ◽  
Interleukin 7 ◽  
Donor Cells

Abstract The precise lineage of dendritic cells (DCs), including skin Langerhans cells (LCs), is unclear. Interleukin 7 (IL-7) and its receptor (IL-7Rα) are known to mediate lymphopoiesis, and IL-7 is also known to be essential for the generation of DCs from lymphoid-committed precursors in vitro. Thus, to determine the developmental lymphoid (or IL-7Rα) dependency of various DCs and to examine the importance of IL-7/IL-7Rα for DC development in vivo, we used IL-7Rα knockout (KO) donor cells to reconstitute DCs/LCs in sublethally irradiated recipients and compared the results to those obtained using wild-type (WT) donor cells. We found that lymphoid lineage cells (except natural killer [NK] cells), including thymocytes, were less efficiently reconstituted by IL-7Rα KO donor cells, whereas myeloid lineage cells and DCs/LCs were equally well reconstituted by both the IL-7Rα KO and WT donor cells. Overall, we conclude that IL-7Rα is not required for the development of DCs/LC in vivo.

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