Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

Cytoskeletal control of centrioles movement during the establishment of polarity in Madin-Darby canine kidney cells.

The two centrioles that are localized close to each other and to the nucleus in single Madin-Darby Canine kidney cells (MDCK) move apart by distances as large as 13 microns after the establishment of extensive cellular junctions. Microfilaments, and possibly microtubules appear to be responsible for this separation. In fully polarized cells, the centrioles are localized just beneath the apical membrane. After disruption of intercellular junctions in low calcium medium, the centrioles move back towards the cell center. This process requires intact microtubules but happens even in the absence of microfilaments. These results indicate that the position of centrioles is determined by opposing forces produced by microtubules and microfilaments and suggest that the balance between these forces is modulated by the assembly of cellular junctions. Centriole separation appears to be an early event in the process that precedes their final positioning in the apical-most region of the polarized cell.

Effect on microtubule dynamics of XMAP230, a microtubule-associated protein present in Xenopus laevis eggs and dividing cells.

The reorganization from a radial [corrected] interphase microtubule (MT) network into a bipolar spindle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD MAP from Xenopus egg extract (XMAP230). This protein is present in eggs, oocytes, testis and a Xenopus tissue culture cell line. It is apparently absent from non-dividing cells in which an immunologically related 200-kD protein is found. XMAP230 is composed of two isoforms with slightly different molecular masses and pIs. It is localized to interphase MTs, dissociates from MTs at the onset of prophase and specifically binds to spindle MTs during metaphase and anaphase. The dissociation constant of XMAP230 is 500 nM, the stoichiometry of binding to MTs is between 1:8 and 1:4, and the in vivo concentration is approximately 200 nM. Both isoforms are phosphorylated and have reduced affinity for microtubules in mitotic extracts. Analysis of the effect of XMAP230 on MT dynamics by video microscopy shows that it increases the growth rate, decreases the shrinking rate of MTs and strongly suppresses catastrophes. These results suggest that in vivo, XMAP230 participates in the control of the MT elongation rate, stabilizes MTs and locally modulates MT dynamics during mitosis.

The core of the mammalian centriole contains gamma-tubulin.

BACKGROUND: The microtubule network, upon which transport occurs in higher cells, is formed by the polymerization of alpha and beta tubulin. The third major tubulin isoform, gamma tubulin, is believed to serve a role in organizing this network by nucleating microtubule growth on microtubule-organizing centers, such as the centrosome. Research in vitro has shown that gamma tubulin must be restored to stripped centrioles to regenerate the centrosomal functions of duplication and microtubule nucleation. RESULTS: We have re-examined the localization of gamma tubulin in isolated and in situ mammalian centrosomes using a novel immunocytochemical technique that preserves antigenicity and morphology while allowing increased accessibility. As expected, alpha tubulin was localized in cytoplasmic and centriolar barrel microtubules and in the associated pericentriolar material. Foci of gamma tubulin were observed at the periphery of the organized pericentriolar material, as reported previously, often near the termini of microtubules. A further and major location of gamma tubulin was a structure within the proximal end of the centriolar barrel. The distributions were complementary, in that alpha tubulin was excluded from the core of the centriole, and gamma tubulin was excluded from the microtubule barrel. CONCLUSIONS: We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.

Detection of decreased susceptibility to fluoroquinolones in Salmonella spp. by five different methods including real-time polymerase chain reaction (PCR).

Detection of Salmonella spp. isolates showing decreased susceptibility to fluoroquinolones has become important owing to the increasing prevalence of these strains and their association with treatment failure. Nalidixic acid agar dilution, nalidixic acid disk diffusion, MicroScan automated system and real-time polymerase chain reaction (PCR) (LightCycler) followed by melting temperature (Tm) analysis are compared with ciprofloxacin agar dilution as suitable methods to detect decreased susceptibility to fluoroquinolones in 100 Salmonella spp. isolates. Three minor discrepancies were found for nalidixic acid disk diffusion, one minor discrepancy was found for nalidixic acid agar dilution and Tm analysis, and one major discrepancy was found for MicroScan. Nalidixic acid disk diffusion was confirmed as a good screening method. Tm analysis is a rapid and accurate method for detecting decreased susceptibility to fluoroquinolones due to gyrA mutations in Salmonella spp.

Tobacco BY-2 cell-free extracts induce the recovery of microtubule nucleating activity of inactivated mammalian centrosomes.

The structure and the molecular composition of the microtubule-organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.

[Nontuberculous mycobacteria in patients with cystic fibrosis]. / Micobacterias ambientales en pacientes adultos con fibrosis quística.

OBJECTIVE: Patients with cystic fibrosis are at great risk of infection by nontuberculous mycobacteria from the environment because of certain predisposing factors such as bronchiectasis, malnutrition, and diabetes. The aim of this study was to analyze the mycobacterial content of sputum smears and cultures from adult patients with cystic fibrosis attended at a specialized unit for adults from March 1997 through December 2001. PATIENTS AND METHODS: Sputum samples were collected prospectively according to a protocol applied at each visit, and during most exacerbations staining and culture for mycobacteria were ordered in addition to the usual cultures for bacteria and fungi. A tuberculin test was performed at the end of the study. RESULTS: Twenty-eight patients (16 men) with cystic fibrosis were enrolled. The mean (SD) age was 25.3 (6.7) years. A total of 251 samples were cultured (range in number of samples per patient, 1-31). The mean period of follow up was 40.3 (22.1) months. The sputum smear was positive in 29 cases (4 patients); the culture was positive in 7 patients. More than 3 samples were positive in only 4 patients. Mycobacterium abscessus was isolated in 3 cases, Mycobacterium avium complex in 2 and Mycobacterium simiae in 1 and other an unidentified rapid growth Mycobacterium species. The Mantoux test was positive in 5 patients. Two of the 4 patients in whose samples mycobacteria were isolated repeatedly required treatment. CONCLUSIONS: The prevalence of nontuberculous mycobacterial infection is high in patients with cystic fibrosis. Staining and culture for mycobacteria should be carried out regularly and whenever exacerbation of pulmonary symptoms cannot be attributed to bacteria usually found in such patients. Patients with recurrent isolations of mycobacteria should be monitored closely.

A new clinical condition linked to a novel mutation in lamins A and C with generalized lipoatrophy, insulin-resistant diabetes, disseminated leukomelanodermic papules, liver steatosis, and cardiomyopathy.

A-Type lamins, arising from the LMNA gene, are intermediate filaments proteins that belong to the lamina, a ubiquitous nuclear network. Naturally occurring mutations in these proteins have been shown to be responsible for several distinct diseases that display skeletal and/or cardiac muscle or peripheral nerve involvement. These include familial partial lipodystrophy of the Dunnigan type and the mandibuloacral dysplasia syndrome. The pathophysiology of this group of diseases, often referred to as laminopathies, remains elusive. We report a new condition in a 30-yr-old man exhibiting a previously undescribed heterozygous R133L LMNA mutation. His phenotype associated generalized acquired lipoatrophy with insulin-resistant diabetes, hypertriglyceridemia, hepatic steatosis, hypertrophic cardiomyopathy with valvular involvement, and disseminated whitish papules. Immunofluorescence microscopic analysis of the patient's cultured skin fibroblasts revealed nuclear disorganization and abnormal distribution of A-type lamins, similar to that observed in patients harboring other LMNA mutations. This observation broadens the clinical spectrum of laminopathies, pointing out the clinical variability of lipodystrophy and the unreported possibility of hypertrophic cardiomyopathy and skin involvement. It emphasizes the fact that the diagnosis of genetic alterations in A-type lamins requires careful and complete clinical and morphological investigations in patients regardless of the presenting signs.

Penicillium brevicompactum as the cause of a necrotic lung ball in an allogeneic bone marrow transplant recipient.

The non-Candida, non-Aspergillus fungal infections are being reported with increasing frequency in BMT patients. One of these agents is Penicillium which has rarely been implicated as a pathogen in these patients. Only a few cases of isolated fungemias have been reported to date. We present the first documented case of invasive lung infection due to Penicillium brevicompactum in an allogeneic BMT recipient. As this case shows, the diagnosis of non-Candida, non-Aspergillus fungal infections may be incorrect if only histologic findings are available, mainly because misdiagnosis with other more common fungus can occur. A positive culture is required in order to make an accurate diagnosis.

Community-acquired urinary tract infection caused by vancomycin-resistant Enterococcus faecalis clinical isolate.

We present a case of urinary tract infection caused by vancomycin-resistant Enterococcus faecalis. The patient is a 62-year-old woman showing no recent admittances. The isolated microorganism was identified by MicroScan (DADE) and API (BioMerieux) and susceptibility was assessed by disk diffusion, E-test and broth microdilution. The isolate was identified as Enterococcus faecalis and showed high MIC for vancomycin (>128 mg/l) and teicoplanin (8 mg/l) but was susceptible to ampicillin. The transmission routes of vancomycin-resistant enterococci in the community and their clinical implications remain uncertain. Healthy carriers have already been described in several countries but this case report represents an unusual finding.

Domain-specific disassembly and reassembly of nuclear membranes during mitosis.

The nuclear envelope contains three distinct membrane domains, the outer membrane, the inner membrane, and the pore membrane, that reversibly vesiculate in mitosis. We previously suggested from single-labeling immunofluorescence microscopy analysis of mitotic cells in culture that mitotic vesiculation of the nuclear membranes may proceed in a domain-specific manner (Chaudhary and Courvalin, J. Cell. Biol. 122, 295-306, 1993). In the present study, we add biochemical support to this hypothesis by sorting domain-specific mitotic vesicles. Antibodies directed against the lamin B receptor, a marker of the inner membrane, and glycoprotein gp210, a marker of the pore membrane, were used to isolate by affinity two populations of mitotic vesicles that were selectively enriched in each of these markers. These two vesicle populations were of different size distribution; the pore membrane-derived vesicles were smaller (80% being < or = 200 nm) than the inner membrane-derived vesicles (80% > or = 200 nm). Double-labeling immunofluorescence microscopy analysis of mitotic cells in culture showed that the time course and topology of disassembly and reassembly of inner and pore membrane domains were different, confirming that domain-specific vesicles are generated during mitosis. In these studies, protein LAP2/thymopoietin beta, another marker of the inner nuclear membrane, was segregating as lamin B receptor, suggesting that both proteins were contained in the same mitotic vesicles.

Dynamics of the nuclear envelope at mitosis and during apoptosis.

The nuclear envelope is a highly dynamic structure that reversibly disassembles and reforms at mitosis. The nuclear envelope also breaks down--irreversibly--during apoptosis, a process essential for development and tissue homeostasis. Analyses of fixed cells, time-lapse, imaging studies of live cells and the development of powerful cell-free extracts derived from gametes or mammalian somatic cells have provided insights on the fate of nuclear envelope proteins during mitosis and apoptosis, and on the mechanisms behind nuclear envelope modifications in these processes. In this review, we discuss evidence leading to our understanding of the dynamics of the nuclear envelope alterations at mitosis and during apoptosis. We also present novel imaging and genetic approaches to the study of nuclear envelope dynamics and function.

Immunolocalization of HP1 proteins in metaphasic mammalian chromosomes.

HP1 proteins are conserved non histone chromosomal proteins involved in the epigenetic repression of transcription. Three HP1 proteins, HP1alpha, HP1beta and HP1gamma are expressed in mammalian cells. Polyclonal antibodies directed against peptides specific for HP1alpha and HP1gamma were elicited in rabbits, affinity purified, then used to localize both proteins on spreads of unfixed metaphasic chromosomes. We show here, by conventional and confocal microscopy, that both proteins are localized at centromeres and pericentromeres, and that HP1gamma is also present on euchromatic sites of chromosome arms.

LBR, a chromatin and lamin binding protein from the inner nuclear membrane, is proteolyzed at late stages of apoptosis.

Chromatin condensation and apposition to the nuclear envelope is an important feature of the execution phase of apoptosis. During this process, lamin proteins that are located between the inner nuclear membrane and heterochromatin are proteolyzed by the apoptosis-specific protease caspase 6. We have investigated the fate of nuclear membranes during apoptosis by studying the lamin B receptor (LBR), a transmembrane protein of the inner nuclear membrane. LBR interacts through its nucleoplasmic amino-terminal domain with both heterochromatin and B-type lamins, and is phosphorylated throughout the cell cycle, but on different sites in interphase and mitosis. We report here that: (i) the amino-terminal domain of LBR is specifically cleaved during apoptosis to generate an approximately 20 kDa soluble fragment; (ii) the cleavage of LBR is a late event of apoptosis and occurs subsequent to lamin B cleavage; (iii) the phosphorylation of LBR during apoptosis is similar to that occurring in interphase. As the association of condensed chromatin with the inner nuclear membrane persists until the late stages of apoptosis, we suggest that the chromatin binding protein LBR plays a major role in maintaining this association.

HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes.

Heterochromatin protein 1 (HP1) is a nonhistone chromosomal protein, first identified in Drosophila, that plays a dose-dependent role in gene silencing. Three orthologs, HP1alpha, HP1beta, and HP1gamma, have been characterized in mammals. While HP1alpha and HP1beta have been unambiguously localized in heterochromatin by immunocytochemical methods, HP1gamma has been found either exclusively associated with euchromatin or present in both euchromatin and heterochromatin. Here, using an antibody directed against a peptide epitope at the carboxyl-terminal end of the molecule, we localize HP1gamma in both euchromatin and heterochromatin compartments of interphase nuclei, as well as in the pericentromeric chromatin and arms of mitotic chromosomes of 3T3 cells. This dual location was also observed in nuclei expressing HP1gamma as a fusion protein with green fluorescent protein. In contrast, when the distribution of HP1gamma was analyzed with antibodies directed against an amino-terminal epitope, the protein was detectable in euchromatin and not in heterochromatin, except for transient heterochromatin staining during the late S phase, when the heterochromatin undergoes replication. These data suggest that the controversial immunolocalization of HP1gamma in chromatin is due to the use of antibodies directed against topologically distinct epitopes, those present at the amino-terminal end of the molecule being selectively masked in nonreplicative heterochromatin.

Ribosomal protein phosphorylation in vivo and in vitro by vaccinia virus.

Ribosomal protein phosphorylation was investigated in Ehrlich ascites tumor cells infected with vaccinia virus (Copenhagen strain). After 90 min of simultaneous infection and 32P-labelling, ribosomal proteins Sa, S2 and S13 appear specifically phosphorylated as well as Sb/La, P1 and S6, which are also phosphorylated in control cells. Sa is an acidic protein, whose phosphorylation has not been observed previously. A kinetic study showed that S2 is phosphorylated very rapidly within 10 min after the beginning of infection and it is complete 1 h later. The phosphorylation of S13 begins after a lag time of about 1 h and is completed after about 2.5 h of infection. Moreover only one phosphate is incorporated into S13 on a serine residue while up to four phosphates are incorporated into S2, the first on a serine and the three following on threonine residues. In vivo experiments, carried out in the presence of cycloheximide and cordycepin, suggest a viral origin for the kinase involved in the phosphorylation of S2 and S13. Moreover, in vitro experiments demonstrated that the kinase associated with the viral cores is capable of phosphorylating S2 on a serine residue only. In our cell/virus system, no significant difference in S6 phosphorylation was detected, when compared to uninfected cells. It is concluded that the specific and efficient phosphorylation of three ribosomal proteins from the 40S ribosomal subunit correlate well with possible translational mechanisms ensuring the efficient expression of early and late genes of vaccinia virus. In the light of these and previous results [Person, A. and Beaud, G. (1986) J. Biol. Chem. 261, 8283-8289], a mechanism is proposed for the shut-off of host protein synthesis and the selective translation of mRNAs of viral origin.

Reconstruction of the centrosome cycle from cryoelectron micrographs.

The absence of detailed in vitro studies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle.

Caspase-dependent proteolysis of integral and peripheral proteins of nuclear membranes and nuclear pore complex proteins during apoptosis.

We have studied the fate of the nuclear envelope (NE) in different human cells committed to apoptosis by different chemical agents. Using a battery of antibodies against marker proteins of the three domains of the nuclear envelope, namely lamin B (LB) for the lamina, transmembrane proteins LBR and LAP2 for the inner nuclear membrane, and nucleoporins p62, Nup153 and gp210 for the nuclear pore complexes (NPCs), we observed a selective and conserved cleavage of LB, LAP2 and Nup153. In lymphoid cells, the rate of cleavage of these markers was independent of the apoptosis inducing agent, actinomycin D or etoposide, and more rapid than in attached epithelial cells. While lamin B is cleaved by caspase 6, the protease responsible for the cleavage of LAP2 and Nup153 was probably caspase 3, since (1) cleavage of both proteins was specifically prevented by in vivo addition of caspase 3 inhibitor Ac-DEVD-CHO and (2) consensus sites for these caspases are present in both proteins. As LB, LAP2 and Nup153 are exposed at the inner face of the nuclear envelope and all interact with chromatin, we suggest that their cleavage allows both the detachment of NE from chromatin and the clustering of NPCs in the plane of the membrane, two conserved morphological features of apoptosis observed in this study.

Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells.

Mammalian heterochromatin proteins 1 (HP1alpha, HP1beta, and HP1gamma) are nonhistone proteins that interact in vitro with a set of proteins that play a role in chromatin silencing, transcription, and chromatin remodeling. Using antibodies specific for each HP1 isoform, we showed that they segregate in distinct nuclear domains of human HeLa cells. By contrast, in mouse 3T3 interphase cells, HP1alpha and HP1beta are strictly colocalized. In mitotic HeLa cells, all of HP1alpha and a fraction of HP1beta and HP1gamma remain associated with chromosomes. Immunostaining of spread HeLa chromosomes showed that HP1alpha is mainly localized on centromeres as shown previously for HP1beta, while HP1gamma is distributed on discrete sites on the arms of chromosomes. Biochemical analysis showed that HP1alpha and HP1gamma are phosphorylated throughout the cell cycle, although more extensively in mitosis than in interphase, while HP1beta apparently remains unphosphorylated. Therefore, despite their extensive sequence conservation, mammalian HP1 isoforms differ widely in their nuclear localization, mitotic distribution and cell cycle-related phosphorylation. Thus, subtle differences in primary sequence and in posttranslational modifications may promote their targeting at different chromatin sites, generating pleiotropic effects.

Enzymatic cycling assay for D-carnitine determination.

An enzymatic method for d-carnitine determination using the enzyme d-carnitine dehydrogenase is described. The assay is based on the amplified signal produced during NAD(+) cycling in the presence of a tetrazolium salt and using phenazine methosulfate as electron carrier. Optimum assay conditions were studied with two tetrazolium salt pairs: 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)/MTT-formazan and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT)/INT-formazan. The first pair (MTT) showed higher sensitivity. The calibration curve was linear from 0.1 to 5 mM d-carnitine, with a quantification limit of 0.1 mM and a relative standard deviation of 1.51%. The procedure is simple, rapid, accurate, and easily automated. It was satisfactorily applied to following d-carnitine levels during the microbial transformation of d-carnitine into l-carnitine and to determining the d-carnitine content of pharmaceutical preparations.