Oscillation modes of microtubules.

Microtubules are long, filamentous protein complexes which play a central role in several cellular physiological processes, such as cell division transport and locomotion. Their mechanical properties are extremely important since they determine the biological function. In a recently published experiment [Phys. Rev. Lett. 89 (2002) 248101], microtubule's Young's and shear moduli were simultaneously measured, proving that they are highly anisotropic. Together with the known structure, this finding opens the way to better understand and predict their mechanical behavior under a particular set of conditions. In the present study, we modeled microtubules by using the finite elements method and analyzed their oscillation modes. The analysis revealed that oscillation modes involving a change in the diameter of the microtubules strongly depend on the shear modulus. In these modes, the correlation times of the movements are just slightly shorter than diffusion times of free molecules surrounding the microtubule. It could be therefore speculated that the matching of the two timescales could play a role in facilitating the interactions between microtubules and MT associated proteins, and between microtubules and tubulins themselves.

Axonemal activity relative to the 2D/3D-waveform conversion of the flagellum.

The waveform of the flagellum of the sea urchin spermatozoon is mainly planar, but its 3D-properties were evoked for dynamic reasons and described as helical. In 1975, the apparent twisting pattern of the sea urchin axoneme was described [Gibbons I. 1975. The molecular basis of flagellar motility in sea urchin spermatozoa. In: Inoué S, Stephens R, editors. Molecular and cellular movement. New York: Raven Press, p. 207-232.] and was considered to be one of the main elements involved in axonemal behaviour. Recently, planar, quasi-planar, and helical waveforms were observed when the flagellum of sea urchin sperm cells was submitted to an increase in viscosity. The quasi-planar conformation seemed to be due to the alternating torsion of the inter-bend segments [Woolley D, Vernon G. 2001. A study of helical and planar waves on sea urchin sperm flagella, with a theory of how they are generated. J. Exp. Biol. 204:1333-1345]. These three waveforms, which are due to a change in axonemal activity, are possibly used by the sperm cells to adapt their movement to variations in the physico-chemical characteristics of the medium (seawater) in which the cells normally swim. We constructed a simple model to describe qualitatively the central shear (between the axonemal doublets and the central pair) and the tangential shear (between the doublets themselves). In this model, the 3D-bending is resolved into components in two perpendicular planes and each of the nine planes of inter-doublet interaction defines a potential bending plane that is independently regulated. These shears were calculated for the three waveforms and their inter-conversion. This allowed us to propose that axoneme is resolved in successive modules delineated by abscissas where the sliding is always nil. We discuss these data concerning the axonemal machinery, and especially the alternating activity of opposite sides of (two) neutral surface(s) that seem(s) to be responsible for this inter-conversion, and for the possible twist of the axoneme during the beating.

Elastic extension and jump of the flagellar nexin links: a theoretical mechanical cycle.

The functions of the nexin links of a flagellar axoneme have not been clearly demonstrated. Taking into account both the elastic (Hookean) characteristics and the possible jump of the nexin links, we calculated the sliding to bending conversion of a theoretical model in a tip-ward direction step by step, according to the essential principles proposed by the geometric clutch hypothesis [Lindemann, 1994: J Theoret Biol 168:175-189]: the activity of the dynein arms depends on the transverse forces induced by the axonemal curvature. In our calculations, however, the transverse forces that are involved in the regulation of the activities of the dynein arms were due to the extension of the nexin links located upstream of a given abscissa. This allowed us to define a bent segment as the axonemal portion at whose proximal and distal ends the nexin links were relaxed, and as fully extended as possible, respectively. The model creates an apparent disorder in the orientation of the nexin links as already observed [Bozkurt and Wooley, 1993: Cell Motil Cytoskeleton 24:109-118; Wooley, 1997: J Cell Sci 110:85-94]. We propose that the nexin links are involved in a mechanical cycle, whose 3 stages are (1) rapid extension, (2) slow relaxation, and (3) stand-by. The rapid extension is compatible with the mechanical interactions between the nexin links and the inner dynein arms with which they form the dynein regulatory complex. This was correlated with the oscillating properties of the nexin links along the axoneme that allow them to be one of the regulatory elements of the local ATPase activity of the dynein arms.

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives.

The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. x P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours.

Effects of osmolality, morphology perturbations and intracellular nucleotide content during the movement of sea bass (Dicentrarchus labrax) spermatozoa.

Sea bass spermatozoa are maintained immotile in the seminal fluid, but initiate swimming for 45 s at 20 degrees C, immediately after dispersion in a hyperosmotic medium (1100 mOsm kg-1). The duration of this motile period could be extended by a reduction of the amplitude of the hyperosmotic shock. Five seconds after the initiation of motility, 94.4 +/- 1.8% of spermatozoa were motile with a swimming velocity of 141.8 +/- 1.2 microns s-1, a flagellar beat frequency of 60 Hz and a symmetric type of flagellar swimming, resulting in linear tracks. Velocity, flagellar beat frequency, percentage of motile cells and trajectory diameter decreased concomitantly throughout the swimming phase. After 30 s of motility, the flagellar beat became asymmetric, leading to circular trajectories. Ca2+ modulated the swimming pattern of demembranated spermatozoa, suggesting that the asymmetric waves produced by intact spermatozoa after 30 s of motility were induced by an accumulation of intracellular Ca2+. Moreover, increased ionic strength in the reactivation medium induced a dampening of waves in the distal portion of the flagellum and, at high values, resulted in an arrest of wave generation in demembranated spermatozoa. In non-demembranated cells, the intracellular ATP concentration fell immediately after transfer to sea water. In contrast, the AMP content increased during the same period, while the ADP content increased slightly. In addition, several morphological changes affected the mitochondria, chromatin and midpiece. These results indicate that the short swimming period of sea bass spermatozoa is controlled by energetic and cytoplasmic ionic conditions and that it is limited by osmotic stress, which induces marked changes in cell morphology.

Calculation of a Gap restoration in the membrane skeleton of the red blood cell: possible role for myosin II in local repair.

Human red blood cells contain all of the elements involved in the formation of nonmuscle actomyosin II complexes (V. M. Fowler. 1986. J. Cell. Biochem. 31:1-9; 1996. Curr. Opin. Cell Biol. 8:86-96). No clear function has yet been attributed to these complexes. Using a mathematical model for the structure of the red blood cell spectrin skeleton (M. J. Saxton. 1992. J. Theor. Biol. 155:517-536), we have explored a possible role for myosin II bipolar minifilaments in the restoration of the membrane skeleton, which may be locally damaged by major mechanical or chemical stress. We propose that the establishment of stable links between distant antiparallel actin protofilaments after a local myosin II activation may initiate the repair of the disrupted area. We show that it is possible to define conditions in which the calculated number of myosin II minifilaments bound to actin protofilaments is consistent with the estimated number of myosin II minifilaments present in the red blood cells. A clear restoration effect can be observed when more than 50% of the spectrin polymers of a defined area are disrupted. It corresponds to a significant increase in the spectrin density in the protein free region of the membrane. This may be involved in a more complex repair process of the red blood cell membrane, which includes the vesiculation of the bilayer and the compaction of the disassembled spectrin network.

The use of phosphocreatine plus ADP as energy source for motility of membrane-deprived trout spermatozoa.

Live trout spermatozoa initiate flagellar motility for a short period of time (30 s at 18 degrees C), during which their mean beat frequency (BF) decreases steadily from 60 to 20 Hz; motility then stops abruptly. When demembranated, the motility of axonemes lasts much longer, up to 20 min, with high beat frequency, provided that ATP (millimolar concentration) and cAMP (micromolar) are added. In the present paper, the motility of demembranated trout sperm was investigated in the absence of added ATP in various incubation conditions relative to other substrates. Without the addition of exogenous creatine kinase, the addition of phosphocreatine (PCr) and ADP shows the appearance of a progressive activation of all sperm models with BF increasing with time up to high values. Without the addition of cAMP, the BF increases to lower values but flagella propagated poorly coordinated waves for only a few min. Similar progressive activation is also observed when only ADP is added (without any previous in vivo activation) and BF increases up to moderate values. In this latter case, no activation occurs without addition of cAMP. The respective roles of creatine kinase and adenylate kinase in this process were investigated by addition of specific inhibitors such as fluorodinitrobenzene and P1,P5-di(adenosine-5')pentaphosphate in the above described conditions. We conclude from these observations that all the elements necessary for a coupling between ADP/PCr/creatine kinase on one hand and ATP/ADP/dynein on the other appear to be present in trout spermatozoa: thus the existence of a shuttle sustaining this coupling is strongly suggested.

Molecular cloning and characterization of a radial spoke head protein of sea urchin sperm axonemes: involvement of the protein in the regulation of sperm motility.

Monoclonal antibodies raised against axonemal proteins of sea urchin spermatozoa have been used to study regulatory mechanisms involved in flagellar motility. Here, we report that one of these antibodies, monoclonal antibody D-316, has an unusual perturbating effect on the motility of sea urchin sperm models; it does not affect the beat frequency, the amplitude of beating or the percentage of motile sperm models, but instead promotes a marked transformation of the flagellar beating pattern which changes from a two-dimensional to a three-dimensional type of movement. On immunoblots of axonemal proteins separated by SDS-PAGE, D-316 recognized a single polypeptide of 90 kDa. This protein was purified following its extraction by exposure of axonemes to a brief heat treatment at 40 degrees C. The protein copurified and coimmunoprecipitated with proteins of 43 and 34 kDa, suggesting that it exists as a complex in its native form. Using D-316 as a probe, a full-length cDNA clone encoding the 90-kDa protein was obtained from a sea urchin cDNA library. The sequence predicts a highly acidic (pI = 4.0) protein of 552 amino acids with a mass of 62,720 Da (p63). Comparison with protein sequences in databases indicated that the protein is related to radial spoke proteins 4 and 6 (RSP4 and RSP6) of Chlamydomonas reinhardtii, which share 37% and 25% similarity, respectively, with p63. However, the sea urchin protein possesses structural features distinct from RSP4 and RSP6, such as the presence of three major acidic stretches which contains 25, 17, and 12 aspartate and glutamate residues of 34-, 22-, and 14-amino acid long stretches, respectively, that are predicted to form alpha-helical coiled-coil secondary structures. These results suggest a major role for p63 in the maintenance of a planar form of sperm flagellar beating and provide new tools to study the function of radial spoke heads in more evolved species.

A functional analysis of p53 during early development of Xenopus laevis.

p53 is a nuclear protein that acts like a tumor suppressor and is involved in regulation of cellular growth. In Xenopus, the p53 protein is highly expressed during oogenesis and is strictly cytoplasmic in the oocyte. We have analysed its participation in DNA replication and transcription during early development, using the egg and oocyte as model-systems. The injection of sperm nuclei into Xenopus eggs is followed by DNA replication and mitotic events. We show that the endogenous p53 enters the nuclei and moves through a series of discrete sub-nuclear loci whose distribution is S-phase specific. A specific peripheral nuclear localization of p53 is observed before entry into S-phase, followed by an internal localization which is strictly dependent on ongoing DNA synthesis. At no stage in the cell cycle, however, did we observe any co-localization with RPA or PCNA, which were used as initiation or elongation markers for DNA replication. We also show that injection into the nucleus of the oocyte of small amounts of either Xenopus or human p53 - less than 10% of the cytoplasmic storage - is sufficient to block RNA polymerase II-dependent transcription from a coinjected TATA-box-containing reporter plasmid. Transcription is rescued by microinjection of the TATA-box binding protein (TBP), suggesting that nuclear exclusion of p53 during oogenesis may be necessary for transcription of maternal genes. These characteristics are discussed in relation to the regulation of nuclear activities during early embryogenesis.

Kinetics of protoporphyrinogen oxidase inhibition by diphenyleneiodonium derivatives.

Protoporphyrinogen oxidase, the last enzyme of the common branch of the heme and chlorophyll pathways in plants, is the molecular target of diphenyl ether-type herbicides. These compounds inhibit the enzyme competitively with respect to the tetrapyrrole substrate, protoporphyrinogen IX. We used the flavinic nature of protoporphyrinogen oxidase to investigate the reactivity of the enzyme toward the 2,2'-diphenyleneiodonium cation, a known inhibitor of several flavoproteins. Diphenyleneiodonium inhibited the membrane-bound yeast protoporphyrinogen oxidase competitively with molecular oxygen. The typical slow-binding kinetics suggested that the enzyme with a reduced flavin rapidly combined with the inhibitor to form an initial complex which then slowly isomerized to a modified enzyme-inhibitor complex (Ki = 6.75 x 10(-8) M, Ki* = 4.1 x 10(-9) M). This inhibition was strongly pH-dependent and was maximal at pH 8. Substituted diphenyleneiodoniums were synthesized and shown to be even better inhibitors than 2,2'-diphenyleneiodonium: Ki = 4.4 x 10(-8) M and Ki* = 1.3 x 10(-9) M for 4-methyl-2,2'-diphenyleneiodonium, Ki = 2.2 x 10(-8) M and Ki * = 1.1 x 10(-9) M for 6-methyl-2,2'-diphenyleneiodonium, and Ki = 6.4 x 10(-9) M and Ki* = 1.2 x 10(-1)2 M for 4-nitro-2,2'-diphenyleneiodonium. The 4-nitro-2,2'-diphenyleneiodonium was a quasi irreversible inhibitor (k5/k6 > 5000). Diphenyleneiodoniums are a new class of protoporphyrinogen oxidase inhibitors that act via a mechanism very different from that of diphenyl ether-type herbicides and appear to be promising tools for studies on the structure-function relationships of this agronomically important enzyme.

Early defect in branching morphogenesis of the ureteric bud in induced nephron deficit.

Development of the metanephric kidney during embryogenesis can be altered both in vivo and in vitro by exposure to gentamicin, which may lead to oligonephronia. To study the role of the ureteric bud in nephron deficit genesis, we used metanephros organ cultures exposed to gentamicin as a model of impaired nephrogenesis. Ultrastructural localization of the antibiotic showed that by eight hours it was already present within the epithelial cells of the ureteric bud and in its growing ends, and also trapped in the adjacent blastema. Using confocal microscopy and image analysis, we devised a quantitative approach to analyze the branching pattern of the ureteric bud, and showed that by 24 hours of culture, despite no change of explants growth, gentamicin had significantly decreased the number of branching points. This effect involved the early branching events and was limited to end buds that had no nephron anlagen nearby. Our findings indicate that impaired branching morphogenesis of the ureteric bud is the likely event of gentamicin-induced nephron deficit.

The polyglutamylated lateral chain of alpha-tubulin plays a key role in flagellar motility.

To investigate whether a specific isotype of tubulin is involved in flagellar motility, we have developed and screened a panel of monoclonal antibodies (mAb) generated against sea urchin sperm axonemal proteins. Antibodies were selected for their ability to block the motility of permeabilized sperm models. The antitubulin mAb B3 completely inhibited, at low concentrations, the flagellar motility of permeabilized sperm models from four sea urchin species. On immunoblots, B3 recognized predominantly alpha-tubulin in sea urchin sperm axonemes and equally well brain alpha- and beta-tubulins. Subtilisin cleavage of tubulin removed the B3 epitope, indicating that it was restricted to the last 13 amino acid residues of the C-terminal domain of alpha-tubulin. In enzyme-linked immunosorbant assays, B3 reacted with glutamylated alpha-tubulin peptides from sea urchin or mouse brain but did not bind to the unmodified corresponding peptide, indicating that it recognized polyglutamylated motifs in the C-terminal domain of alpha-tubulin. On the other hand, other tubulin antibodies directed against various epitopes of the C-terminal domain, with the exception of the antipolyglutamylated mAb GT335, had no effect on motility while having binding properties similar to that of B3. B3 and GT335 acted by decreasing the beating amplitude without affecting the flagellar beat frequency. B3 and GT335 were also capable of inhibiting the motility of flagella of Oxyrrhis marina, a 400,000,000 year old species of dinoflagellate, and those of human sperm models. Localization of the antigens recognized by B3 and GT335 by immunofluorescence techniques revealed their presence along the whole axoneme of sea urchin spermatozoa and flagella of O. marina, except for the distal tip and the cortical microtubule network of the dinoflagellate. Taken together, the data reported here indicate that the polyglutamylated lateral chain of alpha-tubulin plays a dynamic role in a dynein-based motility process.

Inhibition of flagellar beat frequency by a new anti-beta-tubulin antibody.

A panel of monoclonal antibodies (mAbs) has been generated against sea urchin sperm axonemes and selected for their ability to inhibit the motility of sea urchin sperm models. The mAb C9 recognized a 50 kDa protein on blots of sea urchin sperm axonemes. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that C9 recognized isoforms of beta-tubulin. Low concentrations of C9 (0.1-1.0 microgram/ml) blocked the motility of sea urchin sperm models by decreasing the sliding velocity and frequency of flagellar beating to less than 1 Hz and by modifying the shear angle along the axoneme, especially the distal end. Other antitubulin antibodies had little effect on motility at concentrations 100-fold higher than those effective for C9. The effects on motility were not restricted to flagella of sea urchin spermatozoa. Flagellar beating of the dinoflagellate Oxyrrhis marina was completely blocked by C9 in a manner reminiscent of that of sea urchin sperm flagella. The mAb also inhibited the motility of human spermatozoa and Chlamydomonas reinhardtii. Immunofluorescence techniques revealed that C9 stains the whole axoneme of sea urchin spermatozoa and O. marina flagella together with the cortical network of O. marina cell body. C9 is the first antitubulin antibody reported to interfere with flagellar beat frequency. The observation that this antibody arrests the motility of flagella from sea urchin sperm along with that of dinoflagellate, algae, and human sperm flagella suggests that the epitope recognized by C9 is conserved over a long period of evolution and plays an important role in sperm motility.

The 59 kDa FK506-binding protein, a 90 kDa heat shock protein binding immunophilin (FKBP59-HBI), is associated with the nucleus, the cytoskeleton and mitotic apparatus.

FKBP59-HBI, a 59 kDa FK506 binding protein which binds the 90 kDa heat shock protein hsp90 and thus is a heat shock protein binding immunophilin (HBI), was originally discovered in association with unliganded steroid receptors in their heat shock protein containing heterooligomer form. It belongs to a growing family including other FKBPs which bind the immunosuppressants FK506 and rapamycin, and cyclophilins which bind cyclosporin A, all having rotamase (peptidyl-prolyl cis-trans isomerase) activity which may be involved in protein folding. Targets for drug-immunophilin complexes have been mostly studied in vivo in T lymphocytes; however, immunophilins are present in all cell types, where their role and distribution are still unknown. Here we report the localization of FKBP59-HBI in various non lymphoid cells (mouse fibroblasts (L-929), monkey kidney cells (Cos-7), Madin-Darby canine kidney epithelial cells (MDCK), and mouse neuronal cells (GT1)). Two polyclonal antipeptide antibodies directed against the C-terminal end (amino acids 441-458) (Ab 173) or the sequence 182-201 (Ab 790) of the FKBP59-HBI were used in light and confocal laser immunofluorescence. FKBP59-HBI was found in the cytoplasm and nucleus of interphase cells. Specific immunofluorescence was much stronger in the cytoplasm than in the nucleus when using Ab 173, and stronger in the nucleus than in the cytoplasm with Ab 790. Detailed observations of L-cells, which have a particularly flat morphology, showed a punctate as well as a fibrous cytoskeletal staining in the cytoplasm using antibody 173, a result which suggests interactions of FKBP59-HBI with an organized network. Colocalization experiments (using antibodies against tubulin, vimentin or actin) and use of cytoskeletal-disrupting drugs revealed partial association of FKBP59-HBI with the microtubules. Western blot experiments confirmed that the protein was present in the subcellular fractions containing either 'soluble' proteins released from cells exposed to NP40 detergent, or proteins released from the cytoskeleton exposed to calcium ions (i.e. in microtubule depolymerizing conditions). Exposure of cells to 1 microM FK506 and rapamycin for 1 hour did not modify significantly the staining, although rapamycin treatment rendered the network stained by 173 clearly visible. Interestingly, during mitosis FKBP59-HBI segregated from the region of the chromosomes; it mainly localized with the mitotic apparatus (centrosome, spindle and interzone separating the chromosomes), the cleavage furrow and the midbodies during cytokinesis. It appeared again as a fibrous network in the cytoplasm of the two daughters cells.(ABSTRACT TRUNCATED AT 400 WORDS)

The angle of the dorsoventral axis with respect to the anteroposterior axis in the Drosophila embryo is controlled by the distribution of gurken mRNA in the oocyte.

Like most organisms, Drosophila embryos develop in relation to two orthogonal axes, the anteroposterior and dorsoventral. These two axes are established by four independent systems of maternal information. Mutations in any system disrupt either the anteroposterior or the dorsoventral patterning of the embryo but never affect the orthogonal orientation of the axes relative to each other. Here we show by analyzing their embryonic fate map, that K10 embryos still possess a dorsoventral polarity. However, instead of forming a 90 degrees angle, the dorsoventral and the anteroposterior axes lie parallel to each other. This axis misorientation was partially corrected by decreasing the wild-type grk gene copy number such that the embryos issued from K10/K10; grk/+ females showed a variability in their fate map which could be interpreted as a progressive rotation of dorsoventral axis relative to the unmodified anteroposterior axis. This rotation is maximal in the K10 embryos where it reaches 90 degrees and results in the congruence of the two axes. The alteration of the embryonic fate map could be traced back to oogenesis where it was shown to correlate with the mislocalization of the grk transcripts.

The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae.

In order to isolate new mutations impairing transcriptional regulation of sulfur metabolism in Saccharomyces cerevisiae, we used a potent genetic screen based on a gene fusion expressing XylE (from Pseudomonas putida) under the control of the promoter region of MET25. This selection yielded strains mutated in various different genes. We describe in this paper the properties of one of them, MET27. Mutation or disruption of MET27 leads to a methionine requirement and affects S-adenosylmethionine (AdoMet)-mediated transcriptional control of genes involved in sulfur metabolism. The cloning and sequencing of MET27 showed that it is identical to VPS33. Disruptions or mutations of gene VPS33 are well known to impair the biogenesis and inheritance of the vacuolar compartment. However, the methionine requirement of vps33 mutants has not been reported previously. We show here, moreover, that other vps mutants of class C (no apparent vacuoles) also require methionine for growth. Northern blotting experiments revealed that the met27-1 mutation delayed derepression of the transcription of genes involved in sulfur metabolism. By contrast, this delay was not observed in a met27 disrupted strain. Physiological and morphological analyses of met27-1 and met27 disrupted strains showed that these results could be explained by alterations in the ability of the vacuole to transport or store AdoMet, the physiological effector of the transcriptional regulation of sulfur metabolism.

Microtubule tracks can be detected in mouse oocytes with an antibody directed against a calcium transporter.

In metaphase II-arrested mouse oocytes, most microtubules are found in the meiotic spindle, a structure that remains stable for hours despite microtubule instability. Microtubule organizing centres (MTOCs) are present at the poles of the spindle and in the cytoplasm, but the latter nucleate very few microtubules. This particular organization of the microtubule network enabled us to observe the unexpected behaviour of a protein that can associate with microtubules. We compared the distribution of a mitosis-activated calcium transport system with that of the microtubule network, by immunofluorescence, using two monoclonal antibodies, one directed against a component of the calcium transport system (7/13), and the other against the common tyrosinated form of alpha-tubulin (YL1/2). The 7/13 staining was associated with the spindle microtubules and with the kinetochore area. In addition, we observed many asters in the cytoplasm, around the cytoplasmic MTOCs. The majority of these asters were not stained with the antitubulin antibody. Moreover, these 7/13 asters either disappeared after nocodazole treatment or were enlarged after taxol treatment. Using a confocal microscope, we observed single fibres that were stained with both antibodies: the extremity furthest from the MTOC (corresponding to the + end of the microtubule) being detected by the 7/13 antibody only. All these observations suggest that the 7/13 antigen is associated with microtubule tracks that persist a few minutes after microtubule depolymerization. The possible role of these tracks in microtubule regrowth is discussed.

Forskolin blocks the apical expression of dipeptidyl peptidase IV in Caco-2 cells and induces its retention in lamp-1-containing vesicles.

In a previous work, we showed that the differentiation-dependent expression of dipeptidyl peptidase IV (DPP IV) in Caco-2 cells, a human colon adenocarcinoma cell line, was controlled at the mRNA level (D. Darmoul et al. J. Biol. Chem., 1992, 267, 4824-4833). Whether post-translational events may contribute to the final control of DPP IV cell surface expression was explored here by studying the potential effect of forskolin (FK), a drug known to permanently stimulates adenylyl cyclase and to strongly perturbs glucose metabolism in fully differentiated Caco-2 cells. FK treatment reduces by about 50% the amount of active DPP IV present at the brush border membrane, whereas the total amount of active DPP IV remains unchanged. Biosynthesis and maturation of DPP IV were measured using [35S]methionine labeling and were shown to be essentially unaffected by FK treatment. Pulse-chase experiments demonstrate that up to 50% of the neosynthesized DPP IV do not appear at the apical membrane after FK treatment. To get further insight into this phenomenon, we have used confocal laser scanning microscopy. We demonstrate that the blockade of DPP IV transport is associated with the accumulation of this protein in intracellular vesicles. Double-staining experiments demonstrate that these vesicles are not labeled with a monoclonal antibody directed against the Golgi apparatus but display a strong staining with a polyclonal antibody raised against lamp-1, a lysosomal membrane protein. Using a newly developed image analysis procedure, we have been able to quantitate the relative distribution of lamp-1 and DPP IV labels in both control and forskolin-treated cells. We show that the overlap of the two labels dramatically increases in FK-treated Caco-2 cells. These results suggest that, beside the transcriptional level, post-translational events may be involved in the final control of the apical expression of a differentiation-dependent hydrolase.

Okadaic acid induces spindle lengthening and disrupts the interaction of microtubules with the kinetochores in metaphase II-arrested mouse oocytes.

The cell cycle is regulated by phosphorylation events via a cascade of protein kinases and phosphatases, but many of their substrates remain unknown. To study whether proteins of the metaphase II-arrested mouse oocyte meiotic spindle are substrates for phosphorylation events, we used okadaic acid (OA), a potent phosphatase inhibitor, upon fully mature spindles. Incubation of oocytes for 3 hr with 1 microM OA led to a dramatic lengthening of the spindle and a disorganization of the metaphase plate. Electron microscope studies revealed that this was due to a disruption of the interactions between the microtubules and the kinetochores. Biochemical analysis including MPM-2 immunoblotting and [32P]phosphate labeling of whole oocytes revealed several changes in the phosphorylation pattern following the OA treatment. Moreover, meiotic spindle purification or microtubule-associated proteins (MAPs) isolation showed that some of these phosphorylations occur on proteins associated with the microtubules or with structures closely related to the spindle. These results suggest that the changes occurring in the microtubule network during the cell cycle are partly due to the phosphorylation of some proteins associated with the microtubules.

The small GTP-binding protein rab6p is distributed from medial Golgi to the trans-Golgi network as determined by a confocal microscopic approach.

A key role in the regulation of membrane traffic is played by the rab proteins, members of a family of ras-related small GTP-binding proteins. This family comprises at least 25 identified members, the intracellular localization of only a few of which has been investigated. rab6p has been shown to be distributed along the exocytic pathway in association with the medial and trans regions of the Golgi apparatus. A confocal laser scanning microscopic (CLSM) approach coupled with image analysis was used to compare the localization of rab6p with selected reference Golgi markers by double immunofluorescence on culture cell lines. CLSM analysis shows that, under a set of well-defined conditions, one can investigate the possible colocalization of known markers of Golgi compartments and orientate a couple of labeled Golgi antigens with regard to the polarity of the Golgi apparatus. Thus, having validated the CLSM analysis, the localization of rab6p was studied and compared with some of these markers and the VSV-G protein in VSV (vesicular stomatitis virus)-infected cells blocked at 20 degrees C. rab6p is shown to be associated in all the cell lines used with the last cisternae of the Golgi apparatus and particularly with the trans-Golgi network (TGN), the site of protein sorting at the exit of the Golgi apparatus. These results were supported by an electron microscopic study using double-immunolabeled cryosections: rab6p was found in some flat cisternae of the Golgi stack and colocalized with the VSV-G protein in the TGN. Our results show that the small GTP-binding protein rab6p is distributed from medial Golgi to TGN along the exocytic pathway.