Polyglutamine expansion (polyQ) in the protein huntingtin is pathogenic and responsible for the neuronal toxicity associated with Huntington's disease (HD). Although wild-type huntingtin possesses antiapoptotic properties, the relationship between the neuroprotective functions of huntingtin and pathogenesis of HD remains unclear. Here, we show that huntingtin specifically enhances vesicular transport of brain-derived neurotrophic factor (BDNF) along microtubules. Huntingtin-mediated transport involves huntingtin-associated protein-1 (HAP1) and the p150(Glued) subunit of dynactin, an essential component of molecular motors. BDNF transport is attenuated both in the disease context and by reducing the levels of wild-type huntingtin. The alteration of the huntingtin/HAP1/p150(Glued) complex correlates with reduced association of motor proteins with microtubules. Finally, we find that the polyQ-huntingtin-induced transport deficit results in the loss of neurotrophic support and neuronal toxicity. Our findings indicate that a key role of huntingtin is to promote BDNF transport and suggest that loss of this function might contribute to pathogenesis.

No abstract

CLIP-170 is a plus-end tracking protein which may act as an anticatastrophe factor. It has been proposed to mediate the association of dynein/dynactin to microtubule (MT) plus ends, and it also binds to kinetochores in a dynein/dynactin-dependent fashion, both via its C-terminal domain. This domain contains two zinc finger motifs (proximal and distal), which are hypothesized to mediate protein-protein interactions. LIS1, a protein implicated in brain development, acts in several processes mediated by the dynein/dynactin pathway by interacting with dynein and other proteins. Here we demonstrate colocalization and direct interaction between CLIP-170 and LIS1. In mammalian cells, LIS1 recruitment to kinetochores is dynein/dynactin dependent, and recruitment there of CLIP-170 is dependent on its site of binding to LIS1, located in the distal zinc finger motif. Overexpression of CLIP-170 results in a zinc finger-dependent localization of a phospho-LIS1 isoform and dynactin to MT bundles, raising the possibility that CLIP-170 and LIS1 regulate dynein/dynactin binding to MTs. This work suggests that LIS1 is a regulated adapter between CLIP-170 and cytoplasmic dynein at sites involved in cargo-MT loading, and/or in the control of MT dynamics.

The mitotic spindle is often positioned in a characteristic location during development, for example to enable the proper segregation of developmental determinants [1,2]. When epithelial cells divide, the mitotic spindle is often positioned parallel to the plane of the epithelium, so that both daughter cells contribute to the epithelium [3]. The mechanisms by which mitotic spindles are positioned have not been characterized in great detail, but evidence is accumulating that in some systems the dynein-dynactin microtubule motor complex plays a role [4-6]. Dynein has yet not been localized to cortical sites where it could bind to microtubules and exert a force that might orient the mitotic spindle, however [7,8]. Here, we report that in mitotic polarized epithelial cells, the dynein-dynactin complex accumulates, from prometaphase onwards, along astral microtubules and at cortical spots, into which many of the astral microtubules dock. The spots are assembled at the lateral plasma membrane, in the region below the tight junctions. Their formation is inhibited by cytochalasin D, and under these conditions the spindles do not orient properly. This novel localization of the dynein-dynactin complex is consistent with a role for the complex in the positioning of the mitotic spindle. We also show that, during prophase, the motor complex colocalizes with the nuclear envelope, consistent with it having a role in separating the centrosomes that are associated with the nuclear envelope.

In different epithelia, cell membranes contacting one another form intercellular junctional complexes including tight, adherens and gap junctions, which could mutually influence the expression of each other. We have here investigated the role of Cx43 in the control of adherens and tight junction proteins (N-cadherin, beta-catenin, occludin and ZO-1) by using conditional Sertoli cell knockout Cx43 (SCCx43KO(-/-)) transgenic mice and specific anti-Cx43 siRNA. Gap junction coupling and Cx43 levels were reduced in SCCx43KO(-/-) as compared to Wild-type testes. Ultrastructural analysis revealed disappearance of gap junctions, the presence of tight and adherens junctions and persistent integrity of the blood-testis barrier in SCCx43KO(-/-) testis. Occludin, N-cadherin and beta-catenin levels were enhanced in SCCx43KO(-/-) mice as compared to Wild-type animals whereas ZO-1 levels were reduced. Cx43 siRNA blocked gap junction functionality in Sertoli cells and altered tight and adherens protein levels. The Cx43 control of tight and adherens junctions appeared channel-dependent since gap junction blockers (glycyrrhetinic acid and oleamide) led to similar results. These data suggest that the control of spermatogenesis by Cx43 may be mediated through Sertoli cell Cx43 channels, which are required, not only in cell/cell communication between Sertoli and germ cells, but also in the regulation of other junctional proteins essential for the blood-testis barrier.

Cell fusion occurs as part of the differentiation of some cell types, including myotubes in muscle and osteoclasts in remodeling bone. In the human placenta, mononuclear cytotrophoblasts in a human chorionic gonadotropin (hCG)-driven process fuse to form multinucleated syncytia that allow the exchange of nutrients and gases between the maternal and fetal circulation. Experiments in which protein kinase A (PKA) is displaced from A-kinase anchoring proteins (AKAPs), or in which specific AKAPs are depleted by siRNA-mediated knockdown, point to ezrin as a scaffold required for hCG-, cAMP-and PKA-mediated regulation of the fusion process. By a variety of immunoprecipitation and immunolocalization experiments, we show that ezrin directs PKA to a molecular complex of connexin 43 (Cx43, also known as GJA1) and zona occludens-1 (ZO-1, also known as TJP1). A combination of knockdown experiments and reconstitution with ezrin or Cx43 with or without the ability to bind to its interaction partner or to PKA demonstrate that ezrin-mediated coordination of the localization of PKA and Cx43 is necessary for discrete control of Cx43 phosphorylation and hCG-stimulated gap junction communication that triggers cell fusion in cytotrophoblasts.

No abstract

Connexins form gap junction channels that allow intercellular communication between neighboring cells. Compelling evidence has revealed that Cx are tumor-suppressor genes and reduced Cx expression has been related with uncontrolled cell growth in tumors and transformed cells. In the present study, we addressed Cx transcriptional and posttranscriptional regulations during the earlier stage of testicular tumors confined to Leydig cells in a transgenic mice model. In situ hybridization indicated that connexin43 (Cx43) mRNA was highly expressed either at early tumorogenesis (3 m) characterized by intense proliferation of Leydig cells, or at advanced tumorogenesis (6-7 m) when tumor cells completely invaded the testis. In contrast, Cx43 protein analyzed by Western blotting or classic immunohistochemical analyses was present at the beginning of tumor progression, but was dramatically reduced as tumor advanced. Application of high-resolution deconvolution microscopy to testis sections demonstrates that cells that proliferate exhibited an aberrant cytoplasmic Cx43 localization, in contrast to the expected plasma membrane Cx43 localization in normal Leydig cells. Dual immunofluorescence labeling with specific markers of cellular compartments shows that cytoplasmic Cx43 signal was mainly sequestered within early endosomes. Altogether, this study provides the first evidence that impaired Cx43 trafficking in endosomes is an early event associated with uncontrolled cell proliferation that could serve as a neoplastic marker.