Cytoplasmic dynein is the multisubunit motor protein for retrograde movement of diverse cargoes to microtubule minus ends. Here, we investigate the function of dynein variants, defined by different intermediate chain (IC) isoforms, by expressing fluorescent ICs in neuronal cells. Green fluorescent protein (GFP)–IC incorporates into functional dynein complexes that copurify with membranous organelles. In living PC12 cell neurites, GFP–dynein puncta travel in both the anterograde and retrograde directions. In cultured hippocampal neurons, neurotrophin receptor tyrosine kinase B (TrkB) signaling endosomes are transported by cytoplasmic dynein containing the neuron-specific IC-1B isoform and not by dynein containing the ubiquitous IC-2C isoform. Similarly, organelles containing TrkB isolated from brain by immunoaffinity purification also contain dynein with IC-1 but not IC-2 isoforms. These data demonstrate that the IC isoforms define dynein populations that are selectively recruited to transport distinct cargoes.

Mammalian neuroepithelial stem cells divide using a polarized form of cytokinesis, which is not well understood. The cytokinetic furrow cleaves the cell by ingressing from basal to apical, forming the midbody at the apical membrane. The midbody mediates abscission by recruiting many factors, including the Kinesin-6 family member Kif20b. In developing embryos, Kif20b mRNA is most highly expressed in neural stem/progenitor cells. A loss-of-function mutant in Kif20b, magoo, was found in a forward genetic screen. magoo has a small cerebral cortex, with reduced production of progenitors and neurons, but preserved layering. In contrast to other microcephalic mouse mutants, mitosis and cleavage furrows of cortical stem cells appear normal in magoo. However, apical midbodies show changes in number, shape and positioning relative to the apical membrane. Interestingly, the disruption of abscission does not appear to result in binucleate cells, but in apoptosis. Thus, Kif20b is required for proper midbody organization and abscission in polarized cortical stem cells and has a crucial role in the regulation of cerebral cortex growth.

Transcriptional changes of the enzyme nitric oxide synthase I (NOS1) are believed to play a role in the development of many diseases. The gene for NOS1 has 12 alternative first exons (1A-1L). The 1F exon is one of the most highly utilized first exons in the brain and has a polymorphism ((TG)(m)TA(TG)(n)) located in its promoter region. The polymorphism's length has been suggested to affect NOS1 transcription and play a role in Parkinson's disease (PD); however, the actual influence of the polymorphism on NOS1 transcription has not been studied. To better characterize the links of the polymorphism with PD, a genotyping study was done comparing polymorphism length among 170 PD patients and 150 age-matched controls. The pattern of changes between the two group's allele frequencies shows statistical significance (P = 0.0359). The smallest polymorphism sizes are more predominant among PD patients than controls. To study the effects of this polymorphism on NOS1 gene transcription, reporter gene constructs were made by cloning the NOS1 1F promoter with polymorphism lengths of either 42, 54, or 62 bp in front of the luciferase gene and transfecting them into HeLa or Sk-N-MC cells. NOS1-directed reporter gene constructs with the 62-bp polymorphism increased transcription of luciferase 2.2-fold in HeLa and 1.8-fold in Sk-N-MC cells compared with reporter gene constructs with the 42-bp polymorphism. These data suggest that if smaller polymorphism size contributes to the higher NOS1 levels in PD patients, an as yet unknown transcriptional mechanism is required.

Cytoplasmic dynein 1 is a microtubule-based molecular motor that functions to generate force for cargo transport to microtubule minus-ends (1-3). It is involved in numerous eukaryotic cell processes including the trafficking of membranous vesicles, viruses, and other intracellular particles. Cytoplasmic dynein 1 is a large multisubunit complex (ϳ1.5 MDa) containing two copies of six subunits, the heavy chain (DYNC1H), the intermediate chain (DYNC1I), the light intermediate chain (DYNC1LI), and three distinct light chains, DYNLT (previously called Tctex1), DYNLRB (previously called roadblock), and DYNLL (previously called LC8) (2, 4 -6). The motor domains of cytoplasmic dynein 1 are located in the C-terminal globular heads of the two identical heavy chains Whereas there is only a single heavy chain isoform, there are multiple isoforms of the five subunits that make up the cargo binding domain (2,3,17). In mammals, at least six intermediate chain isoforms are produced by the alternative splicing of two genes, and there are at least two genes for each of the other four subunits (2, 3, 5, 18). Assembly of individual subunit isoforms into the dynein complex creates different populations of the motor protein that are thought to be involved in specific cargo binding and regulation (2, 19 -22). For example, pericentrin is transported to the centrosome exclusively by the dynein complexes that contain the light intermediate chain isoform DYNC1LI-1 (23). The DYNLT and DYNLL light chains have been shown to interact with numerous functionally unrelated proteins (3, 14, 15, 24 -27).The two members of the DYNLT family, DYNLT1 (previously called Tctex1) 2 and DYNLT3 (previously called rp3) are found in all cultured cells and adult and fetal tissues so far examined (19,28,29). Unlike the DYNLT isoforms, the expression of the six intermediate chains isoforms are tissue and cell type-specific (18, 30 -32). One intermediate chain isoform, DYNC1I-2C (IC-2C), 3 is found in all cells and it is often the only isoform found in cultured cells (18). Most tissues express only * This work was supported by National Institutes of Health NINDS Grants NS29996 (to K. K. P.) and GM 51293 (to S. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed: P. O. Box 800732, University of Virginia, Charlottesville, VA 22908-0732. Tel.: 434-924-1912; Fax: 434-982-3912; E-mail: kkp9w@virginia.edu.2 Cytoplasmic dynein 1 light chain and intermediate chain subunit nomenclature. There are three functionally distinct light chain families in the cytoplasmic dynein complex and each family has at least two members (or isoforms). The names of the light chains all begin with DYN for dynein, followed by L for light chain, then additional letters that designate the families. The family names are based on the old common name of the first identifie...

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.