Cauda epididymal spermatozoa of the rufous hare wallaby, Lagorchestes hirsutus (Metatheria, Mammalia) imaged by electron and confocal microscopy

The ultrastructure of mature Lagorchestes hirsutus spermatozoa is described for the first time, revealing unusual aspects of sperm structure in macropodid species. The sperm head is ovoid rather than cuneiform, lacks a ventral nuclear groove and has an acrosomal distribution over approximately 85-90% of its dorsal surface. Immediately adjacent to the nuclear membrane the peripheral nucleoplasm in most spermatozoa form an irregular series of distinctive evaginations previously not described in the spermatozoa of any other marsupial. The midpiece is extremely thickened and short, containing no helical network or peripheral plasma membrane specializations. Axonemal structure is unspecialized with no connecting lamellae; dense outer fibres are closely adherent to axonemal doublets. The sperm morphology of this species is highly aberrant in comparison to other macropod taxa and supports the retention of Lagorchestes as a distinctive genus. In light of this new information, skeletal and serological data should be re-evaluated to determine the true taxonomic and phylogenetic position of this species.

Ultrastructure of the mature spermatozoa of caecilians (Amphibia : Gymnophiona)

The spermatozoa of Gymnophiona show the following autapomorphies: 1) penetration of the distal centriole by the axial fiber; 2) presence of an acrosomal baseplate; 3) presence of an acrosome seat (flattened apical end of nucleus); and 4) absence of juxta-axonemal fibers. The wide separation of the plasma membrane bounding the undulating membrane is here also considered to be apomorphic. Three plesiomorphic spermatozoal characters are recognized that are not seen in other Amphibia but occur in basal amniotes: 1) presence of mitochondria with a delicate array of concentric cristae (concentric cristae of salamander spermatozoa differ in lacking the delicate array); 2) presence of peripheral dense fibers associated with the triplets of the distal centriole; and 3) presence of a simple annulus (a highly modified, elongate annulus is present in salamander sperm). The presence of an endonuclear canal containing a perforatorium is a plesiomorphic feature of caecilian spermatozoa that is shared with urodeles, some basal anurans, sarcopterygian fish, and some amniotes. Spermatozoal synapornorphies are identified for 1) the Uraeotyphlidae and Ichthyophiidae, an 2) the Caeciliidae and Typhlonectidae, suggesting that the members of each pair of families are more closely related to each other than to other caecilians. Although caecilian spermatozoa exhibit the clear amphibian synapomorphy of the unilateral location of the undulating membrane and its axial fiber, they have no apomorphic characters that suggest a closer relationship to either the Urodela or Axiura. J. Morphol. 258:179-192, 2003. (C) 2003 Wiley-Liss, Inc.

CCTα and CCTδ chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena

Background - The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTalpha to CCTzeta). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (alpha, beta and gamma) and actin, as quantitatively major substrates. Methodology/Principal findings - We disrupted the genes encoding CCTalpha and CCTdelta subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTalpha or CCTdelta showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTalpha rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTalpha, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTalpha localization in cilia. Conclusions/Significance - Our results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.

Espermiogênese em Eupemphix nattereri (Anura, Leiuperidae): aspectos ultra-estruturais

A maturação dos espermatozóides envolve um extenso e complexo processo que começa com a proliferação e diferenciação das espermatogônias, passa pela meiose e finaliza com a espermiogênese. Nessa fase, eventos envolvendo alterações morfológicas e bioquímicas transformam espermátides em espermatozóides. Aspectos ultra-estruturais da espermiogênese e do espermatozóide do anuro Eupemphix nattereri (Steindachner, 1863) foram analisados através de microscopia eletrônica de transmissão. A espermiogênese envolve condensação da cromatina e alongamento nuclear, com visível eliminação de citoplasma. Nesse estágio, grande quantidade de microtúbulos e glicogênio podem ser visualizados no citoplasma das células de Sertoli, rodeando cada espermátide. O espermatozóide é fusiforme e o acrossomo forma uma capa na região anterior do núcleo. A bainha mitocondrial é encontrada ao redor da porção proximal da cauda. A cauda apresenta o axonema com o modelo 9+2, uma fibra axonemal, a membrana ondulante e ausência de bastão axial. Esta organização apresenta algumas similaridades com espécies do gênero Physalaemus (Leiuperidae) como P. biligonigerus (Cope, 1861), P. gracilis (Boulenger, 1883) e P. fuscomaculatus (Steindachner, 1864).

Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement.

BACKGROUND: Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. AIMS AND METHODS: To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. RESULTS AND CONCLUSIONS: We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.

Kermit interacts with gαo, vang, and motor proteins in Drosophila planar cell polarity.

In addition to the ubiquitous apical-basal polarity, epithelial cells are often polarized within the plane of the tissue - the phenomenon known as planar cell polarity (PCP). In Drosophila, manifestations of PCP are visible in the eye, wing, and cuticle. Several components of the PCP signaling have been characterized in flies and vertebrates, including the heterotrimeric Go protein. However, Go signaling partners in PCP remain largely unknown. Using a genetic screen we uncover Kermit, previously implicated in G protein and PCP signaling, as a novel binding partner of Go. Through pull-down and genetic interaction studies, we find that Kermit interacts with Go and another PCP component Vang, known to undergo intracellular relocalization during PCP establishment. We further demonstrate that the activity of Kermit in PCP differentially relies on the motor proteins: the microtubule-based dynein and kinesin motors and the actin-based myosin VI. Our results place Kermit as a potential transducer of Go, linking Vang with motor proteins for its delivery to dedicated cellular compartments during PCP establishment.

Mechanisms of spindle positioning during "Caenorhabditis elgans" asymmetric cell division

Résumé : Le positionnement correct du fuseau mitotique est crucial pour les divisions cellulaires asymétriques, car il gouverne le contrôle spatial de la division cellulaire et assure la ségrégation adéquate des déterminants cellulaires. Malgré leur importance, les mécanismes contrôlant le positionnement du fuseau mitotique sont encore mal compris. Chez l'embryon au stade une-cellule du nématode Caenorhabditis elegans, le fuseau mitotique est positionné de manière asymétrique durant l'anaphase grâce à l'action de générateurs de force situés au cortex cellulaire, et dont la nature était jusqu'alors indéterminée. Ces générateurs de force corticaux exercent une traction sur les microtubules astraux et sont dépendants de deux protéines Gα et de leurs protéines associées. Cette thèse traite de la nature de la machinerie responsable pour la génération des forces de tractions, ainsi que de son lien avec les protéines Gα et associées. Nous avons combiné des expériences de coupure par faisceau laser du fuseau mitotique avec le contrôle temporel de l'inactivation de gènes ou de l'exposition à des produits pharmacologiques. De cette manière, nous avons établi que la dynéine, un moteur se déplaçant vers l'extrémité négative des microtubules, ainsi que la dynamique des microtubules, sont toutes deux requises pour la génération efficace des forces de tractions. Nous avons démontré que les protéines Gα et leurs protéines associées GPR-1/2 et LIN-5 interagissent in vivo avec LIS-1, un composant du complexe de la dynéine. De plus, nous avons découvert que les protéines Gα, GPR-1/2 et LIN-5 promeuvent la présence du complexe de la dynéine au cortex cellulaire. Nos résultats suggèrent un mécanisme par lequel les protéines Gα permettent le recrutement cortical de GPR-1/2 et LIN-5, assurant ainsi la présence de la dynéine au cortex. Conjointement avec la dynamique des microtubules, ce mécanisme permet la génération des forces de tractions afin d'obtenir une division cellulaire correcte. Comme les mécanismes contrôlant le positionnement du fuseau mitotique et les divisions cellulaires asymétriques sont conservés au cours de l'évolution, nous espérons que les mécanismes élucidés par ce travail sont d'importance générale pour la génération de la diversité cellulaire durant le développement. De plus, ces mécanismes pourraient être applicables à d'autres divisions asymétriques, comme celle des cellules souches, dont le disfonctionnement peut entraîner la génération de cellules cancéreuses. Abstract : Proper spindle positioning is crucial for asymmetric cell division, because it controls spatial aspects of cell division and the correct inheritance of cell-fate determinants. However, the mechanisms governing spindle positioning remain incompletely understood. In the Caenorhabditis elegans one-cell stage embryo, the spindle becomes asymmetrically positioned during anaphase through the action of as-yet unidentified cortical force generators that pull on astral microtubules and that depend on two Gα proteins and associated proteins. This thesis addresses the nature of the force generation machinery and the link with the Gα and associated proteins. By performing spindle-severing experiments following temporally restricted gene inactivation and drug exposure, we established that microtubule dynamics and the minus-end directed motor dynein are both required for generating efficient pulling forces. We discovered that the Gα proteins and their associated proteins GPR-1/2 and LIN-5 interact in vivo with LIS-1, a component of the dynein complex. Moreover, we uncovered that LIN-5, GPR-1/2 and the Gα proteins promote the presence of the dynein complex at the cell cortex. Our findings suggest a mechanism by which the Gα proteins enable GPR-1/2 and LIN-5 recruitment to the cortex, thus ensuring the presence of cortical dynein. Together with microtubule dynamics, this allows pulling forces to be exerted and proper cell division to be achieved. Because the mechanisms of spindle positioning and asymmetric cell division are conserved across evolution, we expect the underlying mechanism uncovered here to be of broad significance for the generation of cell diversity during development. Moreover, this mechanism could be relevant for other asymmetric cell divisions, such as stem cell divisions, whose dysfunction may lead to the generation of cancer cells.

Dominant-Negative Effects of Adult-Onset Huntingtin Mutations Alter the Division of Human Embryonic Stem Cells-Derived Neural Cells.

Mutations of the huntingtin protein (HTT) gene underlie both adult-onset and juvenile forms of Huntington's disease (HD). HTT modulates mitotic spindle orientation and cell fate in mouse cortical progenitors from the ventricular zone. Using human embryonic stem cells (hESC) characterized as carrying mutations associated with adult-onset disease during pre-implantation genetic diagnosis, we investigated the influence of human HTT and of an adult-onset HD mutation on mitotic spindle orientation in human neural stem cells (NSCs) derived from hESCs. The RNAi-mediated silencing of both HTT alleles in neural stem cells derived from hESCs disrupted spindle orientation and led to the mislocalization of dynein, the p150Glued subunit of dynactin and the large nuclear mitotic apparatus (NuMA) protein. We also investigated the effect of the adult-onset HD mutation on the role of HTT during spindle orientation in NSCs derived from HD-hESCs. By combining SNP-targeting allele-specific silencing and gain-of-function approaches, we showed that a 46-glutamine expansion in human HTT was sufficient for a dominant-negative effect on spindle orientation and changes in the distribution within the spindle pole and the cell cortex of dynein, p150Glued and NuMA in neural cells. Thus, neural derivatives of disease-specific human pluripotent stem cells constitute a relevant biological resource for exploring the impact of adult-onset HD mutations of the HTT gene on the division of neural progenitors, with potential applications in HD drug discovery targeting HTT-dynein-p150Glued complex interactions.

Molecular genetics of the immotile short tail sperm defect

Hedelmättömyyttä aiheuttavan siittiöiden puolihäntävian molekyyligenetiikka Suomalaisissa Yorkshire karjuissa yleistyi 1990-luvun lopulla autosomaalisesti ja resessiivisesti periytyvä hedelmättömyyttä aiheuttava siittiöiden puolihäntävika (ISTS, immotile short tail sperm). Sairaus aiheuttaa normaalia lyhyemmän ja täysin liikkumattoman siittiön hännän muodostuksen. Muita oireita sairailla karjuilla ei ole havaittu ja emakot ovat oireettomia. Tämän tutkimuksen tarkoituksena oli kartoittaa siittiöiden puolihäntävian aiheuttava geenivirhe ja kehittää DNA-testi markkeri- ja geeniavusteiseen valintaan. Koko genomin kartoituksessa vian aiheuttava alue paikannettiin sian kromosomiin 16. Paikannuksen perusteella kahden geenimerkin haplotyyppi kehitettiin käytettäväksi markkeri-avusteisessa valinnassa. Sairauteen kytkeytyneen alueen hienokartoitusta jatkettiin geenitestin kehittämiseksi kantajadiagnostiikkaan. Vertailevalla kartoituksella oireeseen kytkeytynyt alue paikannettiin 2 cM:n alueelle ihmisen kromosomiin viisi (5p13.2). Tällä alueella sijaitsevia geenejä vastaavista sian sekvensseistä löydetyn muuntelun perusteella voitiin tarkentaa sairauteen kytkeytyneitä haplotyyppejä. Haplotyyppien perusteella puolihäntäoireeseen kytkeytynyt alue rajattiin kahdeksan geenin alueelle ihmisen geenikartalla. Alueelle paikannetun kandidaattigeenin (KPL2) sekvensointi paljasti introniin liittyneen liikkuvan DNA-sekvenssin, Line-1 retroposonin. Tämä retroposoni muuttaa geenin silmikointia siten, että sitä edeltävä eksoni jätetään pois tai myös osa introni- ja inserttisekvenssiä liitetään geenin mRNA tuotteeseen. Molemmissa tapauksissa tuloksena on lyhentynyt KPL2 proteiini. Tähän retroposoni-inserttiin perustuva geenitesti on ollut sianjalostajien käytössä vuodesta 2006. KPL2 geenin ilmenemisen tarkastelu sialla ja hiirellä paljasti useita kudosspesifisiä silmikointimuotoja. KPL2 geenin pitkä muoto ilmenee pääasiassa vain kiveksessä, mikä selittää geenivirheen aiheuttamat erityisesti siittiön kehitykseen liittyvät oireet. KPL2 proteiinin ilmeneminen hiiren siittiön hännän kehityksen aikana ja mahdollinen yhteistoiminta IFT20 proteiinin kanssa viittaavat tehtävään proteiinien kuljetuksessa siittiön häntään. Mahdollisen kuljetustehtävän lisäksi KPL2 saattaa toimia myös siittiön hännän rakenneosana, koska se paikannettiin valmiin siittiön hännän keskiosaan. Lisäksi KPL2 proteiini saattaa myös toimia Golgin laitteessa sekä Sertolin solujen ja spermatidien liitoksissa, mutta nämä havainnot kuitenkin vaativat lisätutkimuksia. Tämän tutkimuksen tulokset osoittavat, että KPL2 geeni on tärkeä siittiön hännän kehitykselle ja sen rakennemuutos aiheuttaa siittiöiden puolihäntäoireen suomalaisilla Yorkshire karjuilla. KPL2 proteiinin ilmeneminen ja paikannus siittiön kehityksen aikana antaa viitteitä proteiinin toiminnasta. Koska KPL2 geenisekvenssi on erittäin konservoitunut, nämä tulokset tuovat uutta tietoa kaikkien nisäkkäiden siittiöiden kehitykseen ja urosten hedelmättömyyteen syihin.

Desmin: molecular interactions and putative functions of the muscle intermediate filament protein

Desmin is the intermediate filament (IF) protein occurring exclusively in muscle and endothelial cells. There are other IF proteins in muscle such as nestin, peripherin, and vimentin, besides the ubiquitous lamins, but they are not unique to muscle. Desmin was purified in 1977, the desmin gene was characterized in 1989, and knock-out animals were generated in 1996. Several isoforms have been described. Desmin IFs are present throughout smooth, cardiac and skeletal muscle cells, but can be more concentrated in some particular structures, such as dense bodies, around the nuclei, around the Z-line or in costameres. Desmin is up-regulated in muscle-derived cellular adaptations, including conductive fibers in the heart, electric organs, some myopathies, and experimental treatments with drugs that induce muscle degeneration, like phorbol esters. Many molecules have been reported to associate with desmin, such as other IF proteins (including members of the membrane dystroglycan complex), nebulin, the actin and tubulin binding protein plectin, the molecular motor dynein, the gene regulatory protein MyoD, DNA, the chaperone alphaB-crystallin, and proteases such as calpain and caspase. Desmin has an important medical role, since it is used as a marker of tumors' origin. More recently, several myopathies have been described, with accumulation of desmin deposits. Yet, after almost 30 years since its identification, the function of desmin is still unclear. Suggested functions include myofibrillogenesis, mechanical support for the muscle, mitochondrial localization, gene expression regulation, and intracellular signaling. This review focuses on the biochemical interactions of desmin, with a discussion of its putative functions.

Bcl-xL regulation and function in cell cycle checkpoints and progression

Quelques évidences suggèrent que Bcl-xL, un membre anti-apoptotique de la famille Bcl-2, possède également des fonctions au niveau du cycle cellulaire et de ses points-contrôle. Pour étudier la régulation et fonction de Bcl-xL au cours du cycle cellulaire, nous avons généré et exprimé dans des cellules humaines une série de mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser49Ala, Ser56Ala, Ser62Ala et Thr115Ala. L'analyse de cette série de mutants révèle que les cellules exprimant Bcl-xL(Ser62Ala) sont moins stables au point-contrôle G2 du cycle cellulaire comparées aux cellules exprimant le type sauvage ou les autres mutants de phosphorylation incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala et Thr115Ala. Les études de cinétiques de phosphorylation et de localisation de phospho-Bcl-xL(Ser62) dans des cellules synchronisées et suite à l'activation du point-contrôle en G2 médié par l'étoposide (VP16), nous indiquent que phospho-Bcl-xL(Ser62) migre dans les corps nucléolaires durant l'arrêt en G2 dans les cellules exposées au VP16. Une série d'expériences incluant des essais kinase in vitro, l'utilisation d'inhibiteurs pharmacologiques et d'ARN interférant, nous révèlent que Polo kinase 1 (PLK1) et MAPK9/JNK2 sont les protéines kinase impliquées dans la phosphorylation de Bcl-xL(Ser62), et pour son accumulation dans les corps nucléolaires pendant le point-contrôle en G2. Nos résultats indiquent que durant le point-contrôle en G2, phospho-Bcl-xL(Ser62) se lie et se co-localise avec CDK1(CDC2), le complexe cycline-kinase qui contrôle l'entrée en mitose. Nos résultats suggèrent que dans les corps nucléolaires, phospho-Bcl-xL(Ser62) stabilise l'arrêt en G2 en séquestrant CDK1(CDC2) pour retarder l'entrée en mitose. Ces résultats soulignent également que les dommages à l'ADN influencent la composition des corps nucléolaires, structure nucléaire qui émerge maintenant comme une composante importante de la réponse aux dommages à l'ADN. Dans une deuxième étude, nous décrivons que les cellules exprimant le mutant de phosphorylation Bcl-xL(Ser62Ala) sont également plus stables au point-contrôle de l'assemblage du fuseau de la chromatine (SAC) suite à une exposition au taxol, comparées aux cellules exprimant le type sauvage ou d'autres mutants de phosphorylation de Bcl-xL, incluant Thr41Ala, Ser43Ala, Thr47Ala, Ser56Ala. Cet effet est indépendent de la fonction anti-apoptotique de Bcl-xL. Bcl-xL(Ser62) est fortement phosphorylé par PLK1 et MAPK14/SAPKp38α à la prométaphase, la métaphase et à la frontière de l'anaphase, et déphosphorylé à la télophase et la cytokinèse. Phospho-Bcl-xL(Ser62) se trouve dans les centrosomes avec γ-tubuline, le long du fuseau mitotique avec la protéine moteure dynéine et dans le cytosol mitotique avec des composantes du SAC. Dans des cellules exposées au taxol, phospho-Bcl-xL(Ser62) se lie au complexe inhibiteur CDC20/MAD2/BUBR1/BUB3, alors que le mutant Bcl-xL(Ser62Ala) ne se lie pas à ce complexe. Ces résultats indiquent que durant le SAC, la phosphorylation de Bcl-xL(Ser62) accélère la résolution du SAC et l'entrée des cellules en anaphase. Des expériences bloquant l'expression de Bcl-xL révèlent ègalement un taux très élevé de cellules tétraploïdes et binuclées après un traitement au nocodazole, consistant avec une fonction de Bcl-xL durant la mitose et dans la stabilité génomique. Dans la troisième étude, l'analyse fonctionnelle de cette série de mutants de phosphorylation indique également que les cellules exprimant Bcl-xL(Ser49Ala) sont moins stables durant le point-contrôle G2 et entre en cytokinèse plus lentement dans des cellules exposées aux inhibiteurs de la polymérisation/dépolymérisation des tubulines, composantes des microtubules. Ces effets de Bcl-xL(Ser49Ala) sont indépendents de sa fonction anti-apoptotique. La phosphorylation de Bcl-xL(Ser49) est dynamique au cours du cycle cellulaire. Dans des cellules synchronisées, Bcl-xL(Ser49) est phosphorylé en phase S et G2, déphosphorylé à la prométaphase, la métaphase et à la frontière de l'anaphase, et re-phosphorylé durant la télophase et la cytokinèse. Au cours du point-contrôle G2 induit par les dommages à l'ADN, un pool important de phospho-Bcl-xL(Ser49) se trouve aux centrosomes, un site important pour la régulation de l'entrée en mitose. Durant la télophase et la cytokinèse, phospho-Bcl-xL(Ser49) se trouve le long des microtubules avec la protéine moteure dynéine et dans le cytosol mitotique. Finalement, nos résultats suggèrent que PLK3 est responsable de la phosphorylation de Bcl-xL(Ser49), une protéine kinase impliquée pour l'entrée des cellules en mitose et pour la progression de la mitose jusqu'à la division cellulaire.

Structural characterization and distribution of zebrafish germ plasm during interphase and mitosis

In zebrafish, germ cells are responsible for transmitting the genetic information from one generation to the next. During the first cleavages of zebrafish embryonic development, a specialized part of the cytoplasm known as germ plasm, is responsible of committing four blastomeres to become the progenitors of all germ cells in the forming embryo. Much is known about how the germ plasm is spatially distributed in early stages of primordial germ cell development, a process described to be dependant on microtubules and actin. However, little is known about how the material is inherited after it reorganizes into a perinuclear location, or how is the symmetrical distribution regulated in order to ensure proper inheritance of the material by both daughter cells. It is also not clear whether there is a controlled mechanism that regulates the number of granules inherited by the daughter cells, or whether it is a random process. We describe the distribution of germ plasm material from 4hpf to 24hpf in zebrafish primordial germ cells using Vasa protein as marker. Vasa positive material appears to be conglomerate into 3 to 4 big spherical structures at 4hpf. While development progresses, these big structures become smaller perinuclear granules that reach a total number of approximately 30 at 24hpf. We investigated how this transformation occurs and how the minus-end microtubule dependent motor protein Dynein plays a role in this process. Additionally, we describe specific colocalization of microtubules and perinuclear granules during interphase and more interestingly, during all different stages of cell division. We show that distribution of granules follow what seems to be a regulated distribution: during cells division, daughter cells inherit an equal number of granules. We propose that due to the permanent colocalization of microtubular structures with germinal granules during interphase and cell division, a coordinated mechanism between these structures may ensure proper distribution of the material among daughter cells. Furthermore, we show that exposure to the microtubule-depolymerizing drug nocodazole leads to disassembly of the germ cell nuclear lamin matrix, chromatin condensation, and fusion of granules to a big conglomerate, revealing dependence of granular distribution on microtubules and proper nuclear structure.

TNF-alpha mediated transport of NF-kappaB to the nucleus is independent of the cytoskeleton-based transport system in non-neuronal cells

In unstimulated cells, proteins of the nuclear factor kappaB (NF-kappaB) transcription factor family are sequestered in the cytoplasm through interactions with IkappaB inhibitor proteins. Tumor necrosis factor alpha (TNF-alpha) activates the degradation of IkappaB-alpha and the nuclear import of cytoplasmic NF-kappaB. Nuclear localization of numerous cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation. In a former study we have shown that activated NF-kappaB rapidly moves from distal processes in neurons towards the nucleus. The fast transport rate suggests the involvement of motor proteins in the transport of NF-kappaB. Here we address the question how NF-kappaB arrives at the nuclear membrane before import in non-neuronal cells, i.e., by diffusion alone or with the help of active transport mechanisms. Using confocal microscopy imaging and analysis of nuclear protein extracts, we show that NF-kappaB movement through the cytoplasm to the nucleus is independent of the cytoskeleton, in the three cell lines investigated here. Additionally we demonstrate that NF-kappaB p65 is not associated with the dynein/dynactin molecular motor complex. We propose that cells utilize two distinct mechanisms of NF-kappaB transport: (1) signaling via diffusion over short distances in non-neuronal cells and (2) transport via motor proteins that move along the cytoskeleton in neuronal processes where the distances between sites of NF-kappaB activation and nucleus can be vast.

Hsc70 is a novel interactor of NF-kappaB p65 in living hippocampal neurons

Signaling via NF-κB in neurons depends on complex formation with interactors such as dynein/dynactin motor complex and can be triggered by synaptic activation. However, so far a detailed interaction map for the neuronal NF-κB is missing. In this study we used mass spectrometry to identify novel interactors of NF-κB p65 within the brain. Hsc70 was identified as a novel neuronal interactor of NF-κB p65. In HEK293 cells, a direct physical interaction was shown by co-immunoprecipitation and verified via in situ proximity ligation in healthy rat neurons. Pharmacological blockade of Hsc70 by deoxyspergualin (DSG) strongly decreased nuclear translocation of NF-κB p65 and transcriptional activity shown by reporter gene assays in neurons after stimulation with glutamate. In addition, knock down of Hsc70 via siRNA significantly reduced neuronal NF-κB activity. Taken together these data provide evidence for Hsc70 as a novel neuronal interactor of NF-κB p65.

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.