The nuclear lamins function in the regulation of replication, transcription, and epigenetic modifications of chromatin. However, the mechanisms responsible for these lamin functions are poorly understood. We demonstrate that A-and B-type lamins form separate, but interacting, stable meshworks in the lamina and have different mobilities in the nucleoplasm as determined by fluorescence correlation spectroscopy (FCS). [Keywords: Lamins; chromatin; RNA polymerase II transcription; chromosome organization] Supplemental material is available at http://www.genesdev.org. Silencing lamin B1 (LB1) expression dramatically increases the lamina meshwork size and the mobility of nucleoplasmic lamin A (LA). The changes in lamina mesh

Membrane microdomains (lipid rafts) are now recognized as critical for proper compartmentalization of insulin signaling. We previously demonstrated that, in adipocytes in a state of TNF␣-induced insulin resistance, the inhibition of insulin metabolic signaling and the elimination of insulin receptors (IR) from the caveolae microdomains were associated with an accumulation of the ganglioside GM3. To gain insight into molecular mechanisms behind interactions of IR, caveolin-1 (Cav1), and GM3 in adipocytes, we have performed immunoprecipitations, cross-linking studies of IR and GM3, and live cell studies using total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching techniques. We found that (i) IR form complexes with Cav1 and GM3 independently; (ii) in GM3-enriched membranes the mobility of IR is increased by dissociation of the IR-Cav1 interaction; and (iii) the lysine residue localized just above the transmembrane domain of the IR ␤-subunit is essential for the interaction of IR with GM3. Because insulin metabolic signal transduction in adipocytes is known to be critically dependent on caveolae, we propose a pathological feature of insulin resistance in adipocytes caused by dissociation of the IR-Cav1 complex by the interactions of IR with GM3 in microdomains.adipocyte ͉ caveolae microdomain ͉ lipid rafts ͉ live cell imaging ͉ type 2 diabetes

Genome information, which is three-dimensionally organized within cells as chromatin, is searched and read by various proteins for diverse cell functions. Although how the protein factors find their targets remains unclear, the dynamic and flexible nature of chromatin is likely crucial. Using a combined approach of fluorescence correlation spectroscopy, single-nucleosome imaging, and Monte Carlo computer simulations, we demonstrate local chromatin dynamics in living mammalian cells. We show that similar to interphase chromatin, dense mitotic chromosomes also have considerable chromatin accessibility. For both interphase and mitotic chromatin, we observed local fluctuation of individual nucleosomes (~50 nm movement/30 ms), which is caused by confined Brownian motion. Inhibition of these local dynamics by crosslinking impaired accessibility in the dense chromatin regions. Our findings show that local nucleosome dynamics drive chromatin accessibility. We propose that this local nucleosome fluctuation is the basis for scanning genome information.

Alcadeina (Alca) is an evolutionarily conserved type I membrane protein expressed in neurons. We show here that Alca strongly associates with kinesin light chain (K D E4-8 Â10 À9 M) through a novel tryptophan-and aspartic acid-containing sequence. Alca can induce kinesin-1 association with vesicles and functions as a novel cargo in axonal anterograde transport. JNK-interacting protein 1 (JIP1), an adaptor protein for kinesin-1, perturbs the transport of Alca, and the kinesin-1 motor complex dissociates from Alca-containing vesicles in a JIP1 concentration-dependent manner. Alca-containing vesicles were transported with a velocity different from that of amyloid b-protein precursor (APP)-containing vesicles, which are transported by the same kinesin-1 motor. Alca-and APP-containing vesicles comprised mostly separate populations in axons in vivo. Interactions of Alca with kinesin-1 blocked transport of APP-containing vesicles and increased b-amyloid generation. Inappropriate interactions of Alc-and APP-containing vesicles with kinesin-1 may promote aberrant APP metabolism in Alzheimer's disease.

Proteins with expanded polyQ repeats are associated with at least nine neurodegenerative disorders including ataxins 1 and 3, Kennedy's disease, and Huntington's disease (HD) 1,2 . These diseases are dominantly inherited and although the polyQ-containing proteins are expressed widely in the brain, they result in selective neuronal death. There is a significant and striking correlation between the length of the polyQ repeat and pathology; longer repeats result in earlier onset and more severe symptoms with the threshold of approximately 40 glutamine residues. In the case of HD, for example, expanded polyQ in huntingtin (htt) protein causes disease 3,4 . A characteristic of the polyQ diseases is the appearance of neuronal inclusions that are formed by aggregation of the polyQ proteins with other cellular proteins 5,6 . This has led to the "toxic gain-of-function" hypothesis that essential proteins can be sequestered, which 3 over time leads to cellular dysgenesis. The expression of polyQ can cause other metastable proteins to lose functionality, and in turn these proteins amplify the toxicity of polyQ by enhancing overall aggregation 7 . Self-aggregation of polyQ proteins has been proposed to be mediated by association of parallel β-sheet structures 8 . However, the intrinsic in vivo events leading to the aggregation of polyQ proteins remain poorly understood.Protein misfolding is a natural consequence of protein biogenesis. To combat cytotoxicity that results from the accumulation of misfolded proteins, all cells express molecular chaperones that are essential for the productive folding of proteins 9,10 . Molecular chaperones are of several classes; for example, Hsp70/J-domain proteins interact with unfolded or partially folded proteins in concert with co-chaperones, while the chaperone machines of the chaperonin (Hsp60) family form cage-like structures that sequester non-native states of proteins 11 . The chaperonin containing t-complex polypeptide 1 (CCT)/t-complex polypeptide 1 ring complex (TRiC) is a member of chaperonin family 12 that facilitates the folding of proteins in the eukaryotic cytosol upon ATP hydrolysis 13,14 . CCT shows a weak but significant homology to E. coli GroEL and forms a hexadecamer double-troidal complex composed of eight different subunits 15,16 . Substrate proteins are captured in the cavity, and released after folding is completed 17 . Approximately 10% of newly-synthesized proteins have been proposed to be recognized by CCT.Recently, in a genome-wide screen to identify modifier genes for polyQ aggregation in C. elegans, approximately 200 genes were found to be required for the prevention of polyQ aggregation 18 . This included the genes encoding two Hsp70s, one J-protein, and six CCT subunits. These observations suggested a 4 role of CCT in preventing polyQ aggregation. We show here in mammalian cells that CCT has a key protective role against the toxicity of htt/polyQ and affects aggregation process at the soluble stage. In the context of our recent in vitro data showing that ...