ISSN:0886-1544    

DOI:10.1002/cm.970300402

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractIn the ciliated protozoanParamecium, swimming direction is regulated by voltage‐gated Ca2+channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca2+‐unresponsive behavioral mutants ofParamecium, atalantamutants, cannot swim backward even though they have functional Ca2+channels in their ciliary membrane. Severalatalantamutants were characterized with regard to several Ca2+‐dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group,atalantaA (initially characterized by Hinrichsen and Kung [1984:Genet. Res. Camb.43:11–20]), showed a helical swimming path of opposite handedness from that of wild‐type cells when detergent‐permeabilized cells (“models”) were reactivated with MgATP. When cGMP‐dependent protein kinase purified from wild‐type cells was added toatalantaA models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type andatalantamutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutantataA. When ciliary proteins were separated by gel electrophoresis and then phosphorylated “on blot” by purified cGMP‐dependent protein kinase, phosphoprotein patterns were similar in wild type andatamutants except that a 48 kDa protein (p48) fromataA3was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP‐dependent protein kinase purified fromataA3mutant cells. Ciliary p48 may be part of the mechanism that regulates the orientation of the ciliary power str

ISSN:0886-1544    

DOI:10.1002/cm.970300403

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWhile studying cAMP‐dependent dynein α‐heavy chain phosphorylation, we found previously [Stephens and Prior, 1992:J. Cell Sci. 103:999–1012] that high salt extraction of sperm flagella from the musselMytilus edulisor the clamSpisula solidissimaremoved most visible dynein arms, accompanied by an amount of Mg+2‐ATPase that correlated with the mass of dynein α‐and β‐heavy chains removed. However, although almost devoid of ATPase activity, such extracted axonemes retained one third of the heavy chain mass as two sets of electrophoretically‐distinct, vanadate‐cleavable, non‐phosphorylated proteins. To explore the nature of these dynein‐like proteins, antibodies to the α‐ and β‐heavy chains were blot affinity‐purified from a rabbit antiserum raised against gradient‐purifiedSpisula18‐20S flagellar outer arm dynein. Although able to recognize common epitopes of the opposite chain type, neither the α‐nor the β‐heavy chain antibody recognized the tightly‐bound proteins in either species, proving that they are immunologically distinct. While the β‐antibody recognized its heavy chain homolog in gill cilia, the α‐antibody did not, demonstrating immunological distinction between flagellar and ciliary dynein α‐heavy chains. Immunization of a mouse with nitrocellulose strips containing one of the two tightly‐boundSpisulaflagellar proteins produced an antiserum that cross‐reacted with each tightly‐bound protein in both species and also recognized α‐ and β‐heavy chains. The anti‐molluscan serum cross‐reacted strongly with sea urchin sperm flagellar dynein B‐, C‐, and D‐bands, considered to be inner arm components, but not with sea urchin outer arm α‐ or β‐heavy chains. These data indicate that the electro‐phoretically and immunologically distinct, tightly‐bound prote

ISSN:0886-1544    

DOI:10.1002/cm.970300404

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe expression and localization of tensin and cortactin were examined in osteoclast precursors in comparison with isolated osteoclasts on various substrates. Initially, the ability of hen monocytes to differentiate into osteoclasts was evaluated on plastic or glass, and compared to differentiation on bone. Specifically, monocytes were isolated from the medullary bones of egg‐laying hens maintained on a Ca‐deficient diet. Differentiation was monitored morphologically and by quantitation of the ability to form Howship's lacunae in bone slices or resorb radiolabeled bone particles of 20–53 m̈m diameter. These cells differentiated into tartrate resistant acid phosphatase (TRAP)‐positive, bone resorbing, multinucleated syncytia in the presence of cytosine‐1‐β‐D‐arabinofuranoside in a time dependent manner (day 1–6). Differentiation into osteoclast‐like cells was similar whether cultured on plastic, on glass, or on bone. When compared to GAP‐DH control levels, tensin and cortctin mRNA levels increased by 7‐ and 10‐fold, respectively, by day 6. Tensin and cortactin protein levels also increased by 6‐ and 15‐fold, respectively, by day 6. Immunofluorescence of differentiating precursors showed that tensin localized between regions of cell to cell contact and colocalized with vinculin in podosomes of osteoclast‐like cells and of real osteoclasts. Cortactin immunofluorescence was not detectable in monocytes but localized inside tensin/vinculin podosome structures after fusion into osteoclast‐like cells and in freshly isolated osteoclasts. Both tensin and cortactin were associated with attachment complexes used by osteoclast‐like cells and osteoclasts to resorb bone. Specifically, punctate cortactin staining was observed inside tensin staining which formed a double ring structure at the membrane/bone interface of resorbing osteoclasts. These data showed that tensin and cortactin can be used as osteoclast differentiation markers, that participate in attachment complexes used to resorb bone, and that tensin may participate in the fusion process of osteocla

ISSN:0886-1544    

DOI:10.1002/cm.970300405

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractMicrotubule (MT) dynamics vary both spatially and temporally within cells and are thought to be important for proper MT cellular function. Because MT dynamics appear to be closely tied to the guanosine triphosphatase (GTPase) activity of β‐tubulin subunits, we examined the importance of MT dynamics in the budding yeastS. cerevisiaeby introducing aT107Kpoint mutation into a region of the single β‐tubulin gene,TUB2, known to affect the assembly‐dependent GTPase activity of MTs in vitro. Analysis of MT dynamic behavior by video‐enhanced differential interference contrast microscopy, revealed thatT107Ksubunits slowed both the growth rates and catastrophic disassembly rates of individual MTs in vitro. In haploid cellstub2‐T107Kis lethal; but intub2‐T107K/tub2‐590heterozygotes the mutation is viable, dominant, and slows cell‐cycle progression through mitosis, without causing wholesale disruption of cellular MTs. The correlation between the slower growing and shortening rates of MTs in vitro, and the slower mitosis in vivo suggests that MT dynamics are important in budding yeast and may regulate the rate of nuclear movement and segregation. The slower mitosis in mutant celis did not result in premature cytokinesis and cell death, further suggesting that cell‐cycle control mechanisms “sense” the mitotic slowdown, possibly by monitoring MT dynamics directly.

ISSN:0886-1544    

DOI:10.1002/cm.970300406

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA simple procedure for the purification of MAP1B from bovine brain is described. The procedure requires two ion‐exchange chromatographic steps and results in>95% pure MAP1B with a typical recovery of about 25–30 mg/kg of brain tissue. SDS‐PAGE analysis of the purified protein shows that it is composed of a high molecular mass (330kDa) heavy chain and two low molecular mass (32kDa and 18kDa) associated light chains. The estimated stoichiometry of heavy chain:light chain is 1:2 and 1:0.2 mole/mole protein for the 32kDa and 18kDa light chains respectively. Western blotting, using monospecific monoclonal antibodies, shows that only the heavy chain is recognised by the anti‐MAP1B antibody and is not immunostained by either the MAP1A or MAP2 monoclonal antibodies. Purified MAP1B binds efficiently to both unpolymerised tubulin and polymerised tubulin and co‐sediments with taxol‐stabilised microtubules. Co‐incubation experiments show that MAP2 can compete with MAP1B binding to microtubules, indicating common or overlapping sites. However, MAP1B binds to neither G‐actin nor F‐actin nor co‐sediments with F‐actin, suggesting that it is not an

ISSN:0886-1544    

DOI:10.1002/cm.970300407

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA protein component of 62‐kDa (p62) in the mitotic apparatus of the sea urchin embryo has been shown to be important for the proper progression of mitosis [Dinsmore and Sloboda, 1989:Cell 57:127–134]. To study the subcellular distribution of p62 during the cell cycle of sea urchin embryos, indirect immunofluorescence microscopy was used coupled to a modified detergent extraction procedure. The improved fluorescent images obtained by this procedure provide new information concerning the subcellular localization of p62 during the cell cycle that could not be obtained with previous conventional staining procedures [Johnston and Sloboda, 1992:J. Cell Biol.119:843–854]. Using affinity purified antibodies to p62, we observed a cell cycle‐dependent localization of p62 to the chromosomes/chromatin. Prior to nuclear envelope breakdown of the first or second cell cycle, p62 localizes to chromatin in the nucleus. During mitosis, p62 associates with the region of the spindle occupied by the microtubules of the mitotic apparatus. As anaphase proceeds, but before the nuclear envelope reforms, p62 becomes progressively associated with the chromosomes. Thus, p62 is incorporated into the forming interphase nucleus due to its association with chromosomes during late anaphase, rather than by active translocation into the newly formed daughter nuclei through the nuclear pores. The protein is not unique to marine embryos, as demonstrated by immunofluorescence of Y‐1 cells, a mouse adrenal tumor cell line In these cells, the localization of p62 is similar to the localization of the protein in echinoderm embryos, suggesting its possible function in mitotic progression in mammalian somatic cells as well. © 1995 Wiley

ISSN:0886-1544    

DOI:10.1002/cm.970300408

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0886-1544    

DOI:10.1002/cm.970300401

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY