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1.
Functional Dissection and Hierarchy of Tubulin-folding Cofactor Homologues in Fission Yeast 总被引:3,自引:1,他引:2
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Pippa A. Radcliffe Dai Hirata Leah Vardy Takashi Toda 《Molecular biology of the cell》1999,10(9):2987-3001
We describe the isolation of fission yeast homologues of tubulin-folding cofactors B (Alp11) and E (Alp21), which are essential for cell viability and the maintenance of microtubules. Alp11B contains the glycine-rich motif (the CLIP-170 domain) involved in microtubular functions, whereas, unlike mammalian cofactor E, Alp21E does not. Both mammalian and yeast cofactor E, however, do contain leucine-rich repeats. Immunoprecipitation analysis shows that Alp11B interacts with both α-tubulin and Alp21E, but not with the cofactor D homologue Alp1, whereas Alp21E also interacts with Alp1D. The cellular amount of α-tubulin is decreased in both alp1 and alp11 mutants. Overproduction of Alp11B results in cell lethality and the disappearance of microtubules, which is rescued by co-overproduction of α-tubulin. Both full-length Alp11B and the C-terminal third containing the CLIP-170 domain localize in the cytoplasm, and this domain is required for efficient binding to α-tubulin. Deletion of alp11 is suppressed by multicopy plasmids containing either alp21+ or alp1+, whereas alp21 deletion is rescued by overexpression of alp1+ but not alp11+. Finally, the alp1 mutant is not complemented by either alp11+ or alp21+. The results suggest that cofactors operate in a linear pathway (Alp11B-Alp21E-Alp1D), each with distinct roles. 相似文献
2.
Supplying the appropriate amount of correctly folded α/β-tubulin heterodimers is critical for microtubule dynamics. Formation of assembly-competent heterodimers is remarkably elaborate at the molecular level, in which the α- and β-tubulins are separately processed in a chaperone-dependent manner. This sequential step is performed by the tubulin-folding cofactor pathway, comprising a specific set of regulatory proteins: cofactors A–E. We identified the fission yeast cofactor: the orthologue of cofactor C, Tbc1. In addition to its roles in tubulin folding, Tbc1 acts as a GAP in regulating Alp41/Arl2, a highly conserved small GTPase. Of interest, the expression of GDP- or GTP-bound Alp41 showed the identical microtubule loss phenotype, suggesting that continuous cycling between these forms is important for its functions. In addition, we found that Alp41 interacts with Alp1D, the orthologue of cofactor D, specifically when in the GDP-bound form. Intriguingly, Alp1D colocalizes with microtubules when in excess, eventually leading to depolymerization, which is sequestered by co-overproducing GDP-bound Alp41. We present a model of the final stages of the tubulin cofactor pathway that includes a dual role for both Tbc1 and Alp1D in opposing regulation of the microtubule. 相似文献
3.
The biogenesis of microtubules in the cell comprises a series of complex steps, including protein-folding reactions catalyzed by chaperonins. In addition a group of evolutionarily conserved proteins, called cofactors (A to E), is required for the production of assembly-competent alpha-/beta-tubulin heterodimers. Using fission yeast, in which alp11(+), alp1(+), and alp21(+), encoding the homologs for cofactors B, D, and E, respectively, are essential for cell viability, we have undertaken the genetic analysis of alp31(+), the homolog of cofactor A. Gene disruption analysis shows that, unlike the three genes mentioned above, alp31(+) is dispensable for cell growth and division. Nonetheless, detailed analysis of alp31-deleted cells demonstrates that Alp31(A) is required for the maintenance of microtubule structures and, consequently, the proper control of growth polarity. alp31-deleted cells show genetic interactions with mutations in beta-tubulin, but not in alpha-tubulin. Budding yeast cofactor A homolog RBL2 is capable of suppressing the polarity defects of alp31-deleted cells. We conclude that the cofactor-dependent biogenesis of microtubules comprises an essential and a nonessential pathway, both of which are required for microtubule integrity. 相似文献
4.
The proper folding of tubulins and their incorporation into microtubules consist of a series of reactions, in which evolutionarily conserved proteins, cofactors A to E, play a vital role. We have cloned a fission yeast gene (alp41(+)) which encodes a highly conserved small GTP-binding protein homologous to budding yeast CIN4 and human ARF-like Arl2. alp41(+) is essential, disruption of which results in microtubule dysfunction and growth polarity defects. Genetic analysis indicates that Alp41 plays a crucial role in the cofactor-dependent pathway, in which it functions upstream of the cofactor D homologue Alp1(D) and possibly in concert with Alp21(E). 相似文献
5.
Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast. 总被引:10,自引:1,他引:9
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The main structural components of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of assembly-competent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein. 相似文献
6.
Andrey Smertenko Yaroslav Blume Vladimír Viklický Zdeněk Opatrný Pavel Dráber 《Planta》1997,201(3):349-358
Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin
variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were
detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts.
They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated
and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin
provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and
Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge
variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin
molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies
against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against
nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally
modified and that these modifications participate in the generation of plant tubulin polymorphism.
Received: 2 May 1996 / Accepted: 16 September 1996 相似文献
7.
Deepsharan K. Dhani Benjamin T. Goult Gifty M. George Daniel T. Rogerson Danny A. Bitton Crispin J. Miller John W. R. Schwabe Kayoko Tanaka 《Molecular biology of the cell》2013,24(21):3337-3349
In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64–amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3Alp6. Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3Alp6. 相似文献
8.
Xiaoming Yang Hongchang Li X. Shawn Liu Anping Deng Xiaoqi Liu 《The Journal of biological chemistry》2009,284(42):28775-28782
CLIP-170, the founding member of microtubule “plus ends tracking” proteins, is involved in many critical microtubule-related functions, including recruitment of dynactin to the microtubule plus ends and formation of kinetochore-microtubule attachments during metaphase. Although it has been reported that CLIP-170 is a phosphoprotein, neither have individual phosphorylation sites been identified nor have the associated kinases been extensively studied. Herein, we identify Cdc2 as a kinase that phosphorylates CLIP-170. We show that Cdc2 interacts with CLIP-170 mediating its phosphorylation on Thr287 in vivo. Significantly, expression of CLIP-170 with a threonine 287 to alanine substitution (T287A) results in its mislocalization, accumulation of Plk1 and cyclin B, and block of the G2/M transition. Finally, we found that depletion of CLIP-170 leads to centrosome reduplication and that Cdc2 phosphorylation of CLIP-170 is required for the process. These results demonstrate that Cdc2-mediated phosphorylation of CLIP-170 is essential for the normal function of this protein during cell cycle progression.Microtubule dynamics consist of alternating phases of growth and shortening, a pattern of behavior known as dynamic instability (1). This process is tightly regulated by a group of proteins that bind specifically to the plus ends of the growing microtubules (2). Cytoplasmic linker protein (CLIP)3 -170, the founding member of the microtubule plus end family (3), is composed of three separate regions: N terminus, central coiled-coil region, and C terminus. In addition to two conserved cytoskeleton-associated protein glycine-rich (CAP-Gly) domains, the N terminus has three serine-rich regions. The N-terminal domain plays an essential role in microtubule targeting (4), the long central coiled-coil domain is responsible for dimerization of the protein, and the C-terminal region, which contains two zinc-finger domains interferes with microtubule binding by interacting with the N terminus (5). Experiments in a variety of organisms have demonstrated that CLIP-170 plays an important role in microtubule dynamics (6, 7). In addition to its positive role in regulating microtubule growth in both yeast and humans (8, 9), CLIP-170 is involved in recruitment of dynactin to the microtubule plus ends and in linking microtubules to the cortex through Cdc42 and IQGAP (10, 11). The role of CLIP-170 during mitosis was recently examined by loss-of-function approaches. It was shown that CLIP-170 localizes to unattached kinetochores in prometaphase and that such localization is essential for the formation of kinetochore-microtubule attachments (12, 13).It was previously reported that CLIP-170 is a phosphoprotein and that overall phosphorylation of CLIP-170 affects its microtubule binding ability (14). More recently, metabolic labeling experiments indicated that CLIP-170 is phosphorylated at multiple sites (15). However, individual phosphorylation sites have not been identified. Moreover, the FKBP12-rapamycin-associated protein (FRAP) is the only kinase identified to date for CLIP-170 (15). Therefore, to fully understand the regulation of CLIP-170, it is important to identify individual phosphorylation sites and the responsible kinases. In this communication, we describe a novel kinase/substrate partnership between Cdc2 and CLIP-170. We provide evidence that Cdc2 phosphorylates CLIP-170 at Thr287, and the Cdc2-mediated phosphorylation of CLIP-170 is essential for its localization at microtubule plus ends in the G2 phase and the G2/M transition. 相似文献
9.
Peter Bieling Stefanie Kandels-Lewis Ivo A. Telley Juliette van Dijk Carsten Janke Thomas Surrey 《The Journal of cell biology》2008,183(7):1223-1233
The microtubule cytoskeleton is crucial for the internal organization of eukaryotic cells. Several microtubule-associated proteins link microtubules to subcellular structures. A subclass of these proteins, the plus end–binding proteins (+TIPs), selectively binds to the growing plus ends of microtubules. Here, we reconstitute a vertebrate plus end tracking system composed of the most prominent +TIPs, end-binding protein 1 (EB1) and CLIP-170, in vitro and dissect their end-tracking mechanism. We find that EB1 autonomously recognizes specific binding sites present at growing microtubule ends. In contrast, CLIP-170 does not end-track by itself but requires EB1. CLIP-170 recognizes and turns over rapidly on composite binding sites constituted by end-accumulated EB1 and tyrosinated α-tubulin. In contrast to its fission yeast orthologue Tip1, dynamic end tracking of CLIP-170 does not require the activity of a molecular motor. Our results demonstrate evolutionary diversity of the plus end recognition mechanism of CLIP-170 family members, whereas the autonomous end-tracking mechanism of EB family members is conserved. 相似文献
10.
The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in
contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC50 greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC50 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both
P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction
between taxol and fungal microtubules, binding studies were performed with fungal cells, using [3H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [3H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient
binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized
taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin.
Received: 16 March 1999 / Accepted: 21 August 1999 相似文献
11.
C. L. BOKROS J. D. HUGDAHL S. S. D. BLUMENTHAL L. C. MOREJOHN 《Plant, cell & environment》1996,19(5):539-548
To obtain information on plant microtubule stability to low temperature and Ca2+, the regulatory domain of polymerized tubulin from maize (Zea mays ev. Black Mexican Sweet) was dissected by limited proteolysis with subtilisin. Tubulin in taxol-stabilized microtubules was cleaved in a subtilisin concentration- and time-dependent manner. Immunoblotting of microtubules with antibodies having mapped epitopes on α- and β-tubulins revealed that cleavage initially removed ≤15 residues from the β-tubulin carboxyl terminus to produce αβs-microtubules. Subsequent cleavage occurred at an extreme site and an internal site within the α-tubulin carboxyl terminus. Electron microscopy revealed that αβs-microtubules were ultra structurally indistinguishable from uncleaved control αβ-micro-tubules. Quantitative polymer sedimentation showed that low temperature treatment (0°C) caused significant depolymerization of αβ-microtubules, but little depolymerization of αβs-microtubules. Ca2+ enhanced the cold-induced depolymerization of both αβ- and αβs-microtubules. However, αβs-microtubules were significantly more stable to depolymerization by cold and Ca2+ than were αβ-micro-tubules. The results showed that maize microtubules containing shortened β-tubulin carboxyl termini are relatively resistant to the combined depolymerizing effects of cold and Ca2+. Thus, the extreme carboxyl terminus of β-tubulin is a crucial element of the plant tubulin regulatory domain and may be involved in the modulation of microtubule stability during the chilling response in plants. 相似文献
12.
《Cell cycle (Georgetown, Tex.)》2013,12(9):1246-1253
Bipolar spindle assembly is essential to genomic stability in dividing cells. Centrosomes or spindle pole bodies duplicated earlier at G1/S remain adjacent until triggered at mitotic onset to become bipolar. Pole reorientation is stabilized by microtubule interdigitation but mechanistic details for bipolarity remain incomplete. To investigate the contribution of spindle pole microtubule organizing center (MTOC) proteins in bipolarity, we applied genetic, structural and molecular biochemical analysis along with timelapse microscopy. Spindle formation was followed by an in vivo growth assay with the conditional allele cut7-22ts, encoding fission yeast mitotic Kinesin-5, essential for bipolarity. By analysis of double and triple mutant strains of MTOC alleles and cut7-22ts we found that stabilized microtubules or increased bundling can rescue cut7-22ts associated bipolarity defects. These changes to microtubule dynamics and organization occurred through two surface domains on γ-tubulin, a helix 11 domain and an adjacent site for binding MTOC protein Alp4. We demonstrate that Kinesin-14 Pkl1, known to oppose bipolarity, can bind to γ-tubulin at helix 11 and that mutation of either of two conserved residues in helix 11 can impair Kinesin-14 binding. Altering the Alp4/γ-tubulin interaction, conserved residues in helix 11 or deletion of pkl1 each are sufficient to rescue bipolarity in our cut7-22ts strain. Our findings provide novel insights into regulation of the bipolar mechanism through the MTOC complex. 相似文献
13.
A fourth component of the fission yeast gamma-tubulin complex,Alp16, is required for cytoplasmic microtubule integrity and becomes indispensable when gamma-tubulin function is compromised
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gamma-Tubulin functions as a multiprotein complex, called the gamma-tubulin complex (gamma-TuC), and composes the microtubule organizing center (MTOC). Fission yeast Alp4 and Alp6 are homologues of two conserved gamma-TuC proteins, hGCP2 and hGCP3, respectively. We isolated a novel gene, alp16(+), as a multicopy suppressor of temperature-sensitive alp6-719 mutants. alp16(+) encodes a 759-amino-acid protein with two conserved regions found in all other members of gamma-TuC components. In addition, Alp16 contains an additional motif, which shows homology to hGCP6/Xgrip210. Gene disruption shows that alp16(+) is not essential for cell viability. However, alp16 deletion displays abnormally long cytoplasmic microtubules, which curve around the cell tip. Furthermore, alp16-deleted mutants are hypersensitive to microtubule-depolymerizing drugs and synthetically lethal with either temperature-sensitive alp4-225, alp4-1891, or alp6-719 mutants. Overproduction of Alp16 is lethal, with defective phenotypes very similar to loss of Alp4 or Alp6. Alp16 localizes to the spindle pole body throughout the cell cycle and to the equatorial MTOC at postanaphase. Alp16 coimmunoprecipitates with gamma-tubulin and cosediments with the gamma-TuC in a large complex (>20 S). Alp16 is, however, not required for the formation of this large complex. We discuss evolutional conservation and divergence of structure and function of the gamma-TuC between yeast and higher eukaryotes. 相似文献
14.
Interdependency of fission yeast Alp14/TOG and coiled coil protein Alp7 in microtubule localization and bipolar spindle formation 总被引:3,自引:0,他引:3
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Sato M Vardy L Angel Garcia M Koonrugsa N Toda T 《Molecular biology of the cell》2004,15(4):1609-1622
The Dis1/TOG family plays a pivotal role in microtubule organization. In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation. Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles. Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles. Alp14 localization to these sites is fully dependent upon Alp7. Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles. Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7. Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization. Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14. Alp7 may be a functional homologue of animal TACC. Our results shed light on an interdependent relationship between Alp14/TOG and Alp7. We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules. 相似文献
15.
Kamlesh K. Gupta Aranda R. Slabbekoorn Benjamin A. Paulson Holly V. Goodson 《Journal of molecular biology》2010,395(5):1049-774
Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network. 相似文献
16.
17.
The fission yeast gamma-tubulin complex is required in G(1) phase and is a component of the spindle assembly checkpoint
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Microtubule polymerization is initiated from the microtubule organizing centre (MTOC), which contains the gamma-tubulin complex. We have identified fission yeast Alp4 and Alp6, which are homologues of the gamma-tubulin-interacting proteins Sc.Spc97/Hs.Gcp2 and Sc. Spc98/Hs.Gcp3, respectively. The size of the fission yeast gamma-tubulin complex is large (>2000 kDa), comparable to that in metazoans. Both Alp4 and Alp6 localize to the spindle pole body (SPB) and also to the equatorial MTOC. Temperature-sensitive (ts) alp4 and alp6 mutants show two types of microtubular defects. First, monopolar mitotic spindles form. Secondly, abnormally long cytoplasmic microtubules appear that do not stop at the cell tips and are still associated with the SPB. Alp4 function is required in G(1) phase and ts mutants become lethal before S-phase. alp4 and alp6 mutants are hypersensitive to the microtubule- destabilizing drug thiabendazole (TBZ) and show a lethal 'cut' phenotype in its presence. Furthermore, alp4mad2 double mutants show an exaggerated multiple septation phenotype in TBZ. These results indicate that Alp4 and Alp6 may play a crucial role in the spindle pole-mediated checkpoint pathway. 相似文献
18.
Shiels H O'Connell A Qureshi MA Howarth FC White E Calaghan S 《Molecular and cellular biochemistry》2007,294(1-2):173-180
Cardiac microtubule stability is increased in the streptozotocin (STZ) model of type 1 diabetes. Here, we investigate the
reason for increased microtubule stability, and the functional consequences of stable microtubule disruption. Ventricular
myocytes were isolated from rats at 8–12 weeks after injection of STZ. A 10% increase in microtubule density, but no difference
in the ratio of microtubule-associated protein 4 (MAP4) to tubulin was seen in myocytes from STZ rats. Functionally, STZ myocytes
showed a tendency for reduced shortening and intracellular Ca2+ ([Ca2+]
i
) transient amplitude, and a significant prolongation of time to peak (ttp) shortening and [Ca2+]
i
. Although microtubules in STZ myocytes were less sensitive to the microtubule disruptor nocodazole (NOC; 33 μM) than control
myocytes, we only saw marked functional consequences of microtubule disruption by NOC in myocytes from diabetic animals. NOC
increased shortening and [Ca2+]
i
transient amplitude in STZ myocytes by 45 and 24%, respectively (compared with 4 and 6% in controls). Likewise, NOC decreased
ttp shortening and [Ca2+]
i
only in STZ myocytes, such that these parameters were no longer different between the two groups. In conclusion, stable microtubules
in diabetes are not associated with an increase in MAP4, but are functionally relevant to cardiac dysfunction in diabetes,
regulating both [Ca2+]
i
and shortening.
Holly Shiels and Anthony O’Connell are equal first authorship. 相似文献
19.
Catterou M Dubois F Schaller H Aubanelle L Vilcot B Sangwan-Norreel BS Sangwan RS 《Planta》2001,212(5-6):673-683
In order to elucidate the involvement of brassinosteroids in the cell elongation process leading to normal plant morphology,
indirect immunofluorescence and molecular techniques were use to study the expression of tubulin genes in the bul1-1 dwarf mutant of Arabidopsis thaliana (L.) Heynh., the characteristics of which are reported in this issue (M. Catterou et al., 2001). Microtubules were studied
specifically in the regions of the mutant plant where the elongation zone is suppressed (hypocotyls and petioles), making
the reduction in cell elongation evident. Indirect immunofluorescence of α-tubulin revealed that very few microtubules were
present in mutant cells, resulting in the total lack of the parallel microtubule organization that is typical of elongating
cells in the wild type. After brassinosteroid treatment, microtubules reorganized and became correctly oriented, suggesting
the involvement of brassinosteroids in microtubule organization. Molecular analyses showed that the microtubule reorganization
observed in brassinosteroid-treated bul1-1 plants did not result either from an activation of tubulin gene expression, or from an increase in tubulin content, suggesting
that a brassinosteroid-responsive pathway exists which allows microtubule nucleation/organization and cell elongation without
activation of tubulin gene expression.
Received: 28 April 2000 / Accepted: 6 October 2000 相似文献