Live imaging of marked chromosome regions reveals their dynamic resolution and compaction in mitosis.

When human cells enter mitosis, chromosomes undergo substantial changes in their organization to resolve sister chromatids and compact chromosomes. To comprehend the timing and coordination of these events, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. Here we achieved this by analyzing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatids cycle between nonresolved and partially resolved states with an interval of a few minutes during G2 phase before completing full resolution in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase while local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of condensin II and I activities, which differentially promote sister chromatid resolution and chromosome compaction, respectively. Our assay reveals novel aspects of dynamics in mitotic chromosome resolution and compaction that were previously obscure in global chromosome assays.

mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for cell cycle exit and myogenic differentiation.

Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Previously we have shown that the mTORC1 signaling inhibitor, RAD001, decreased protein synthesis rates, delayed C2C12 myoblast differentiation, decreased p70S6K activity but did not affect the hypermodification of 4E-BP1. Here we have further investigated the modification of 4E-BP1 during the early phase of differentiation as cells exit the cell cycle, using inhibitors to target mTOR kinase and siRNAs to ablate the expression of raptor and rictor. As predicted, inhibition of mTOR kinase activity prevented p70S6K, 4E-BP1 phosphorylation and was associated with an inhibition of myogenic differentiation. Surprisingly, extensive depletion of raptor did not affect p70S6K or 4E-BP1 phosphorylation, but promoted an increase in mTORC2 activity (as evidenced by increased Akt Ser473 phosphorylation). These data suggest that an mTOR kinase-dependent, but raptor-independent regulation of downstream signaling is important for myogenic differentiation.

Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane.

Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype-phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the Tecta(L1820F,G1824D/+) mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the Tecta(C1837G/+) mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the Tecta(C1619S/+) mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8-40 kHz range are elevated by 30-40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20-30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

mRNA encoding WAVE-Arp2/3-associated proteins is co-localized with foci of active protein synthesis at the leading edge of MRC5 fibroblasts during cell migration.

During cell spreading, mammalian cells migrate using lamellipodia formed from a large dense branched actin network which produces the protrusive force required for leading edge advancement. The formation of lamellipodia is a dynamic process and is dependent on a variety of protein cofactors that mediate their local regulation, structural characteristics and dynamics. In the present study, we show that mRNAs encoding some structural and regulatory components of the WAVE [WASP (Wiskott-Aldrich syndrome protein) verprolin homologous] complex are localized to the leading edge of the cell and associated with sites of active translation. Furthermore, we demonstrate that steady-state levels of ArpC2 and Rac1 proteins increase at the leading edge during cell spreading, suggesting that localized protein synthesis has a pivotal role in controlling cell spreading and migration.

Localization of ribosomes and translation initiation factors to talin/beta3-integrin-enriched adhesion complexes in spreading and migrating mammalian cells.

BACKGROUND INFORMATION: The spatial localization of translation can facilitate the enrichment of proteins at their sites of function while also ensuring that proteins are expressed in the proximity of their cognate binding partners. RESULTS: Using human embryonic lung fibroblasts and employing confocal imaging and biochemical fractionation techniques, we show that ribosomes, translation initiation factors and specific RNA-binding proteins localize to nascent focal complexes along the distal edge of migrating lamellipodia. 40S ribosomal subunits appear to associate preferentially with beta3 integrin in focal adhesions at the leading edges of spreading cells, with this association strongly augmented by a synergistic effect of cell engagement with a mixture of extracellular matrix proteins. However, both ribosome and initiation factor localizations do not require de novo protein synthesis. CONCLUSIONS: Taken together, these findings demonstrate that repression, complex post-transcriptional regulation and modulation of mRNA stability could potentially be taking place along the distal edge of migrating lamellipodia.

Lack of galectin-1 results in defects in myoblast fusion and muscle regeneration.

Galectin-1 has been implicated in the development of skeletal muscle, being maximally expressed at the time of myofiber formation. Furthermore, in the presence of exogenous galectin-1, mononuclear myoblasts show increased fusion in vitro. In the current study, we have used the galectin-1 null mouse to elucidate the role of galectin-1 in skeletal muscle development and regeneration. Myoblasts derived from the galectin-1 mutant showed a reduced ability to fuse in vitro. In galectin-1 null mutants, there was evidence of a delay in muscle fiber development at the neonatal stage and muscle fiber diameter was reduced when compared with wild-type at the adult stage. Muscle regeneration was also compromised in the galectin-1 mutant with the process being delayed and a reduced fiber size being maintained. These results, therefore, show a definitive role for galectin-1 in fusion of myoblasts both in vitro, in vivo, and in regeneration after recovery from induced injury.