Microtubule-dependent cell morphogenesis in the fission yeast.

In many systems, microtubules contribute spatial information to cell morphogenesis, for instance in cell migration and division. In rod-shaped fission yeast cells, microtubules control cell morphogenesis by transporting polarity factors, namely the Tea1-Tea4 complex, to cell tips. This complex then recruits the DYRK kinase Pom1 to cell ends. Interestingly, recent work has shown that these proteins also provide long-range spatial cues to position the division site in the middle of the cell and temporal signals to coordinate cell length with the cell cycle. Here I review how these microtubule-associated proteins form polar morphogenesis centers that control and integrate both spatial and temporal aspects of cell morphogenesis.

Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

The control of microtubule stability in vitro and in transfected cells by MAP1B and SCG10.

In neurons, the regulation of microtubules plays an important role for neurite outgrowth, axonal elongation, and growth cone steering. SCG10 family proteins are the only known neuronal proteins that have a strong destabilizing effect, are highly enriched in growth cones and are thought to play an important role during axonal elongation. MAP1B, a microtubule-stabilizing protein, is found in growth cones as well, therefore it was important to test their effect on microtubules in the presence of both proteins. We used recombinant proteins in microtubule assembly assays and in transfected COS-7 cells to analyze their combined effects in vitro and in living cells, respectively. Individually, both proteins showed their expected activities in microtubule stabilization and destruction respectively. In MAP1B/SCG10 double-transfected cells, MAP1B could not protect microtubules from SCG10-induced disassembly in most cells, in particular not in cells that contained high levels of SCG10. This suggests that SCG10 is more potent to destabilize microtubules than MAP1B to rescue them. In microtubule assembly assays, MAP1B promoted microtubule formation at a ratio of 1 MAP1B per 70 tubulin dimers while a ratio of 1 SCG10 per two tubulin dimers was needed to destroy microtubules. In addition to its known binding to tubulin dimers, SCG10 binds also to purified microtubules in growth cones of dorsal root ganglion neurons in culture. In conclusion, neuronal microtubules are regulated by antagonistic effects of MAP1B and SCG10 and a fine tuning of the balance of these proteins may be critical for the regulation of microtubule dynamics in growth cones.

Selective changes in the neurofilament and microtubule cytoskeleton of NF-H/LacZ mice.

This study focused mainly on changes in the microtubule cytoskeleton in a transgenic mouse where beta-galactosidase fused to a truncated neurofilament subunit led to a decrease in neurofilament triplet protein expression and a loss in neurofilament assembly and abolished transport into neuronal processes in spinal cord and brain. Although all neurofilament subunits accumulated in neuronal cell bodies, our data suggest an increased solubility of all three subunits, rather than increased precipitation, and point to a perturbed filament assembly. In addition, reduced neurofilament phosphorylation may favor an increased filament degradation. The function of microtubules seemed largely unaffected, in that tubulin and microtubule-associated proteins (MAP) expression and their distribution were largely unchanged in transgenic animals. MAP1A was the only MAP with a reduced signal in spinal cord tissue, and differences in immunostaining in various brain regions corroborate a relationship between MAP1A and neurofilaments.

Birth and rapid subcellular adaptation of a hominoid-specific CDC14 protein

Gene duplication was prevalent during hominoid evolution, yet little is known about the functional fate of new ape gene copies. We characterized the CDC14B cell cycle gene and the functional evolution of its hominoid-specific daughter gene, CDC14Bretro. We found that CDC14B encodes four different splice isoforms that show different subcellular localizations (nucleus or microtubule-associated) and functional properties. A microtubular CDC14B variant spawned CDC14Bretro through retroposition in the hominoid ancestor 18-25 million years ago (Mya). CDC14Bretro evolved brain-/testis-specific expression after the duplication event and experienced a short period of intense positive selection in the African ape ancestor 7-12 Mya. Using resurrected ancestral protein variants, we demonstrate that by virtue of amino acid substitutions in distinct protein regions during this time, the subcellular localization of CDC14Bretro progressively shifted from the association with microtubules (stabilizing them) to an association with the endoplasmic reticulum. CDC14Bretro evolution represents a paradigm example of rapid, selectively driven subcellular relocalization, thus revealing a novel mode for the emergence of new gene function

Surfactant protein A, exposure to endotoxin, and asthma in garbage collectors and in wastewater workers.

Endotoxin causes an inflammation at the bronchial and alveolar level. The inflammation-induced increase in permeability of the bronchoalveolar epithelial barrier is supposed to cause a leakage of pneumoproteins. Therefore, their concentrations are expected to increase in the bloodstream.This study aimed at examining the association between occupational exposure to endotoxin and a serum pneumoprotein, surfactant protein A, to look for nonoccupational factors capable of confounding this association, and examine the relation between surfactant protein A and spirometry. There were 369 control subjects, 325 wastewater workers, and 84 garbage collectors in the study. Exposure to endotoxin was assessed through personal sampling and the Limulus amebocytes lysate assay. Surfactant protein A was determined by an in house sandwich enzyme-linked immunosorbent assay (ELISA) in 697 subjects. Clinical and smoking history were ascertained and spirometry carried out according to American Thoracic Society criteria. Multiple linear regression was used for statistical analysis. Exposure was fairly high during some tasks in wastewater workers but did not influence surfactant protein A. Surfactant protein A was lower in asthmatics. Interindividual variability was large. No correlation with spirometry was found. Endotoxin has no effect on surfactant protein A at these endotoxin levels and serum surfactant protein A does not correlate with spirometry. The decreased surfactant protein A secretion in asthmatics requires further study.

The ubiquitin-like protein LC3 regulates the Rho-GEF activity of AKAP-Lbc.

AKAP-Lbc is a member of the A-kinase anchoring protein (AKAP) family that has been recently associated with the development of pathologies, such as cardiac hypertrophy and cancer. We have previously demonstrated that, at the molecular level, AKAP-Lbc functions as a guanine nucleotide exchange factor (GEF) that promotes the specific activation of RhoA. In the present study, we identified the ubiquitin-like protein LC3 as a novel regulatory protein interacting with AKAP-Lbc. Mutagenesis studies revealed that LC3, through its NH(2)-terminal alpha-helical domain, interacts with two binding sites located within the NH(2)-terminal regulatory region of AKAP-Lbc. Interestingly, LC3 overexpression strongly reduced the ability of AKAP-Lbc to interact with RhoA, profoundly impairing the Rho-GEF activity of the anchoring protein and, as a consequence, its ability to promote cytoskeletal rearrangements associated with the formation of actin stress fibers. Moreover, AKAP-Lbc mutants that fail to interact with LC3 show a higher basal Rho-GEF activity as compared with the wild type protein and become refractory to the inhibitory effect of LC3. This suggests that LC3 binding maintains AKAP-Lbc in an inactive state that displays a reduced ability to promote downstream signaling. Collectively, these findings provide evidence for a previously uncharacterized role of LC3 in the regulation of Rho signaling and in the reorganization of the actin cytoskeleton.

The evolutionary conserved BER1 gene is involved in microtubule stability in yeast

In yeast, microtubules are dynamic filaments necessary for spindle and nucleus positioning, as well as for proper chromosome segregation. We identify a function for the yeast gene BER1 (Benomyl REsistant 1) in microtubule stability. BER1 belongs to an evolutionary conserved gene family whose founding member Sensitivity to Red light Reduced is involved in red-light perception and circadian rhythms in Arabidopsis. Here, we present data showing that the ber1Delta mutant is affected in microtubule stability, particularly in presence of microtubule-depolymerising drugs. The pattern of synthetic lethal interactions obtained with the ber1Delta mutant suggests that Ber1 may function in N-terminal protein acetylation. Our work thus suggests that microtubule stability might be regulated through this post-translational modification on yet-to-be determined proteins

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR V 45; and J 45; segment selection as well as CDR3 45; composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Triiodothyronine and nerve growth factor are required to induce cytoplasmic dynein expression in rat dorsal root ganglion cultures.

Beside the several growth factors which play a crucial role in the development and regeneration of the nervous system, thyroid hormones also contribute to the normal development of the central and peripheral nervous system. In our previous work, we demonstrated that triiodothyronine (T3) in physiological concentration enhances neurite outgrowth of primary sensory neurons in cultures. Neurite outgrowth requires microtubules and microtubule associated proteins (MAPs). Therefore the effects of exogenous T3 or/and nerve growth factors (NGF) were tested on the expression of cytoskeletal proteins in primary sensory neurons. Dorsal root ganglia (DRG) from 19 day old rat embryos were cultured under four conditions: (1) control cultures in which explants were grown in the absence of T3 and NGF, (2) cultures grown in the presence of NGF alone, (3) in the presence of T3 alone or (4) in the presence of NGF and T3 together. Analysis of proteins by SDS-polyacrylamide gel electrophoresis revealed the presence of several proteins in the molecular weight region around 240 kDa. NGF and T3 together induced the expression of one protein, in particular, with a molecular weight above 240 kDa, which was identified by an antibody against MAP1c, a protein also known as cytoplasmic dynein. The immunocytochemical detection confirmed that this protein was expressed only in DRG explants grown in the presence of NGF and T3 together. Neither control explants nor explants treated with either NGF or T3 alone expressed dynein. In conclusion, a combination of nerve growth factor and thyroid hormone is necessary to regulate the expression of cytoplasmic dynein, a protein that is involved in retrograde axonal transport.

Clic4, a novel protein that sensitizes 46;-cells to apoptosis.

OBJECTIVES: Chloride intracellular channel protein 4 (Clic4) is a ubiquitously expressed protein involved in multiple cellular processes including cell-cycle control, cell differentiation, and apoptosis. Here, we investigated the role of Clic4 in pancreatic 46;-cell apoptosis. METHODS: We used 46;TC-tet cells and islets from 46;-cell specific Clic4 knockout mice ( 46;Clic4KO) and assessed cytokine-induced apoptosis, Bcl2 family protein expression and stability, and identified Clic4-interacting proteins by co-immunoprecipitation and mass spectrometry analysis. RESULTS: We show that cytokines increased Clic4 expression in 46;TC-tet cells and in mouse islets and siRNA-mediated silencing of Clic4 expression in 46;TC-tet cells or its genetic inactivation in islets 46;-cells, reduced cytokine-induced apoptosis. This was associated with increased expression of Bcl-2 and increased expression and phosphorylation of Bad. Measurement of Bcl-2 and Bad half-lives in 46;TC-tet cells showed that Clic4 silencing increased the stability of these proteins. In primary islets 46;-cells, absence of Clic4 expression increased Bcl-2 and Bcl-xL expression as well as expression and phosphorylation of Bad. Mass-spectrometry analysis of proteins co-immunoprecipitated with Clic4 from 46;TC-tet cells showed no association of Clic4 with Bcl-2 family proteins. However, Clic4 co-purified with proteins from the proteasome suggesting a possible role for Clic4 in regulating protein degradation. CONCLUSIONS: Collectively, our data show that Clic4 is a cytokine-induced gene that sensitizes 46;-cells to apoptosis by reducing the steady state levels of Bcl-2, Bad and phosphorylated Bad.

PKA antagonizes CLASP-dependent microtubule stabilization to re-localize Pom1 and buffer cell size upon glucose limitation.

Cells couple growth with division and regulate size in response to nutrient availability. In rod-shaped fission yeast, cell-size control occurs at mitotic commitment. An important regulator is the DYRK-family kinase Pom1, which forms gradients from cell poles and inhibits the mitotic activator Cdr2, itself localized at the medial cortex. Where and when Pom1 modulates Cdr2 activity is unclear as Pom1 medial cortical levels remain constant during cell elongation. Here we show that Pom1 re-localizes to cell sides upon environmental glucose limitation, where it strongly delays mitosis. This re-localization is caused by severe microtubule destabilization upon glucose starvation, with microtubules undergoing catastrophe and depositing the Pom1 gradient nucleator Tea4 at cell sides. Microtubule destabilization requires PKA/Pka1 activity, which negatively regulates the microtubule rescue factor CLASP/Cls1/Peg1, reducing CLASP's ability to stabilize microtubules. Thus, PKA signalling tunes CLASP's activity to promote Pom1 cell side localization and buffer cell size upon glucose starvation.

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

Regulation of the memory T cell development and islet beta-cell survival by DAPK-related apoptosis-inducing protein kinase 2 (Drak2)

Drak2 est un membre de la famille des protéines associées à la mort et c’est une sérine/thréonine kinase. Chez les souris mutantes nulles Drak2, les cellules T ne présentent aucune défectuosité apparente en apoptose induite par activation, après stimulation avec anti-CD3 et anti-CD28, mais ont un seuil de stimulation réduit, comparées aux cellules T de type sauvage (TS). Dans notre étude, l’analyse d’hybridation in situ a révélé que l’expression de Drak2 est ubiquiste au stade de la mi-gestation chez les embryons, suivie d’une expression plus focale dans les divers organes pendant la période périnatale et l’âge adulte, notamment dans le thymus, la rate, les ganglions lymphatiques, le cervelet, les noyaux suprachiasmatiques, la glande pituitaire, les lobes olfactifs, la médullaire surrénale, l’estomac, la peau et les testicules. Nous avons créé des souris transgéniques (Tg) Drak2 en utilisant le promoteur humain beta-actine. Ces souris Tg montraient des ratios normaux entre cellules T versus B et entre cellules CD4 versus CD8, mais leur cellularité et leur poids spléniques étaient inférieurs comparé aux souris de type sauvage. Après activation TCR, la réponse proliférative des cellules T Tg Drak2 était normale, même si leur production d’interleukine (IL)-2 et IL-4 mais non d’interféron-r était augmentée. Les cellules T Tg Drak2 activées ont démontré une apoptose significativement accrue en présence d’IL-2 exogène. Au niveau moléculaire, les cellules T Tg Drak2 ont manifesté une augmentation moins élevée des facteurs anti-apoptotiques durant l’activation; un tel changement a probablement rendu les cellules vulnérables aux attaques subséquentes d’IL-2. L’apoptose compromise dans les cellulesT Tg Drak2 a été associée à un nombre réduit de cellules T ayant le phénotype des cellules mémoires (CD62Llo) et avec des réactions secondaires réprimées des cellules T dans l’hypersensibilité de type différé. Ces résultats démontrent que Drak2 s’exprime dans le compartiment des cellules T mais n’est pas spécifique aux cellules T; et aussi qu’il joue des rôles déterminants dans l’apoptose des cellules T et dans le développement des cellules mémoires T. En outre, nous avons recherché le rôle de Drak2 dans la survie des cellules beta et le diabète. L’ARNm et la protéine Drak2 ont été rapidement induits dans les cellules beta de l’îlot après stimulation exogène par les cytokines inflammatoires ou les acides gras libres et qui est présente de façon endogène dans le diabète, qu’il soit de type 1 ou de type 2. La régulation positive de Drak2 a été accompagnée d’une apoptose accrue des cellules beta. L’apoptose des cellules beta provoquée par les stimuli en question a été inhibée par la chute de Drak2 en utilisant petit ARNi. Inversement, la surexpression de Drak2 Tg a mené à l’apoptose aggravée des cellules beta déclenchée par les stimuli. La surexpression de Drak2 dans les îlots a compromis l’augmentation des facteurs anti-apoptotiques, tels que Bcl-2, Bcl-xL et Flip, sur stimulation par la cytokine et les acides gras libres. De plus, les expériences in vivo ont démontré que les souris Tg Drak2 étaient sujettes au diabète de type 1 dans un modèle de diabète provoqué par de petites doses multiples de streptozotocine et qu’elles étaient aussi sujettes au diabète de type 2 dans un modèle d’obésité induite par la diète. Nos données montrent que Drak2 est défavorable à la survie des cellules beta. Nous avons aussi étudié la voie de transmission de Drak2. Nous avons trouvé que Drak2 purifiée pouvait phosphoryler p70S6 kinase dans une analyse kinase in vitro. Lasurexpression de Drak2 dans les cellules NIT-1 a entraîné l’augmentation de la phosphorylasation p70S6 kinase tandis que l’abaissement de Drak2 dans ces cellules a réduit la phosphorylation. Ces recherches mécanistes ont prouvé que p70S6 kinase était véritablement un substrat de Drak2 in vitro et in vivo. Cette étude a découvert les fonctions importantes de Drak2 dans l’homéostasie des cellules T et le diabète. Nous avons prouvé que p70S6 kinase était un substrat de Drak2. Nos résultats ont approfondi nos connaissances de Drak2 à l’intérieur des systèmes immunitaire et endocrinien. Certaines de nos conclusions, comme les rôles de Drak2 dans le développement des cellules mémoires T et la survie des cellules beta pourraient être explorées pour des applications cliniques dans les domaines de la transplantation et du diabète.

Microtubule disrupting N-phenyl-N’-(2-chloroethyl) ureas display anticancer activity on cell adhesion, P-glycoprotein and BCL-2-mediated drug resistance.

Objective: Our research program has focused on the development of promising, soft alkylating N-phenyl-N’-(2-chloroethyl)urea (CEU) compounds which acylate the glutamic acid-198 of β-tubulin, near the binding site of colchicum alkaloids. CEUs inhibit the motility of cancerous cells in vitro and, interestingly, exhibit antiangiogenic and anticancer activity in vivo. Mitotic arrest induced by microtubule-interfering agents such as CEUs remains the major mechanism of their anticancer activity, leading to apoptosis. However, we recently demonstrated that microtubule disruption by CEUs and other common antimicrotubule agents greatly alters the integrity and organization of microtubule-associated structures, the focal adhesion contact, thereby initiating anoikis, an apoptosis-like cell death mechanism caused by the loss of cell contact with the extracellular matrix. Methods: To ascertain the activated signaling pathway profile of CEUs, flow cytometry, Western blot, immunohistochemistry and transfection experiments were performed. Wound-healing and chick embryo assays were carried out to evaluate the antiangiogenic potency of CEUs. Results: CEU-induced apoptosis involved early cell cycle arrest in G2/M and increased level of CDK1/cycline B proteins. These signaling events were followed by the specific activation of the intrinsic apoptosis pathway, involving loss of mitochondrial membrane potential (Δψm) and ROS production, cytochrome c release from mitochondria, caspase activation, AIF nuclear translocation, PARP cleavage and nuclear fragmentation. CEUs maintained their efficacy on cells plated on pro-survival extracellular matrices or exhibiting overexpression of P-glycoprotein or the anti-apoptotic protein Bcl-2. Conclusion: Our results suggest that CEUs represent a promising new class of antimicrotubule, antiangiogenic and pro-anoikis agents.