Kinetochore alignment within the metaphase plate is regulated by centromere stiffness and microtubule depolymerases

During mitosis in most eukaryotic cells, chromosomes align and form a metaphase plate halfway between the spindle poles, about which they exhibit oscillatory movement. These movements are accompanied by changes in the distance between sister kinetochores, commonly referred to as breathing. We developed a live cell imaging assay combined with computational image analysis to quantify the properties and dynamics of sister kinetochores in three dimensions. We show that baseline oscillation and breathing speeds in late prometaphase and metaphase are set by microtubule depolymerases, whereas oscillation and breathing periods depend on the stiffness of the mechanical linkage between sisters. Metaphase plates become thinner as cells progress toward anaphase as a result of reduced oscillation speed at a relatively constant oscillation period. The progressive slowdown of oscillation speed and its coupling to plate thickness depend nonlinearly on the stiffness of the mechanical linkage between sisters. We propose that metaphase plate formation and thinning require tight control of the state of the mechanical linkage between sisters mediated by centromeric chromatin and cohesion

Palaeomagnetic and mineral magnetic analyses of the Deckenschotter of northern Switzerland and southern Germany

The Deckenschotter is a fluvial to glaciofluvial gravel unit in northern Switzerland and southern Germany. The deposits are considered the oldest preserved glacial to interglacial Quaternary deposits in the northern Alpine foreland and are thus important geomorphological markers for landscape evolution. Nevertheless, the age of the deposits is only approximately known and subject to controversial debates. This study presents the results of an extensive palaeomagnetic investigation carried out on intercalated fine-grained sediments at 11 sites of the Höhere Deckenschotter (HDS) and at 5 sites of the Tiefere Deckenschotter (TDS). The HDS show reversed and normal magnetisations, indicating deposition > 0.773 Ma, while the TDS exhibit only normal directions. Age constraints for the different sites are discussed in the light of evidence from other studies. The study therefore clearly supports the efforts to determine the age of the Deckenschotter. As data from previous palaeomagnetic studies on the HDS and TDS have not been published or preserved, this is in fact the only data-based palaeomagnetic study available

X-linked microtubule-associated protein, Mid1, regulates axon development

Opitz syndrome (OS) is a genetic neurological disorder. The gene responsible for the X-linked form of OS, Midline-1 (MID1), encodes an E3 ubiquitin ligase that regulates the degradation of the catalytic subunit of protein phosphatase 2A (PP2Ac). However, how Mid1 functions during neural development is largely unknown. In this study, we provide data from in vitro and in vivo experiments suggesting that silencing Mid1 in developing neurons promotes axon growth and branch formation, resulting in a disruption of callosal axon projections in the contralateral cortex. In addition, a similar phenotype of axonal development was observed in the Mid1 knockout mouse. This defect was largely due to the accumulation of PP2Ac in Mid1-depleted cells as further down-regulation of PP2Ac rescued the axonal phenotype. Together, these data demonstrate that Mid1-dependent PP2Ac turnover is important for normal axonal development and that dysregulation of this process may contribute to the underlying cause of OS

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

The product of the von Hippel-Lindau tumor suppressor gene stabilizes microtubules by inhibiting GTPase activity

Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin- based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load- free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation

Formation of talin-vinculin pre-complexes dictates maturation of nascent adhesions by accelerated force transmission and vinculin recruitment

Talin, vinculin, and paxillin are mechanosensitive proteins that are recruited early to nascent integrin-based adhesions (NAs). Using machine learning, high-resolution traction force microscopy, single-particle-tracking and fluorescence fluctuation time-series analysis, we find that, only in the NAs that eventually mature to focal adhesions, all three molecules are recruited concurrently and in synchrony with force onset. Thereafter, vinculin assembles at ~5 fold higher rates than in non-maturing NAs. We identify a domain in talin, R8, which exposes a vinculin- binding-site (VBS) without requiring tension. Stabilizing this domain via mutation lowers tension- free vinculin binding in conjunction with talin, impairs maturation of NAs, and reduces the rate of additional vinculin recruitment after force onset. Taken together, our data show that talin forms a complex with vinculin, before association with integrins, which is essential for NA maturation by talin’s effective unfolding and exposure of additional VBSs that induce fast force growth and further vinculin binding

Identification and characterization of short-chain dehydrogenase/reductase 3 (DHRS3) deficiency, a retinoic acid embryopathy of humans

Purpose Signaling by the morphogen all-trans-retinoic acid (RA) is critical for embryonic development, during which its tissue concentration must be tightly regulated. We investigated eight sibships (12 individuals) segregating five different homozygous variants of DHRS3, which encodes an embryonically expressed enzyme (short-chain dehydrogenase/reductase 3; also termed SDR16C1) that catalyses the reduction of retinaldehyde to retinol, limiting excessive RA synthesis. Methods We assessed variant pathogenicity using comparative phenotypic and bioinformatic analysis, quantification of DHRS3 expression, and measurement of plasma retinoid metabolites. Results Five homozygotes from three families (one family segregating a deletion of the promoter and 5′-untranslated region (UTR) of DHRS3, the other two a missense variant p.(Val171Met)), manifested a congruent phenotype including coronal craniosynostosis, dysmorphic facial features, congenital heart disease (4/5 individuals) and scoliosis (5/5 individuals). Transcription of DHRS3 in whole blood cells from two homozygotes for the promoter/5′-UTR deletion was 90-98% reduced. Cells transfected with a DHRS3-Val171Met construct exhibited reduced retinaldehyde reduction capacity compared to wild-type, yielding reduced retinol and elevated RA; correspondingly, plasma from homozygous patients had significantly reduced retinol and elevated RA (exceeding the normal range), compared to controls and heterozygous relatives. Three additional homozygous missense variants of DHRS3 [p.(Val110Ile), p.(Gly115Asp), p.(Glu244Gln)] were shown to reduce catalytic activity in vitro and/or in vivo, but were associated with normal or different phenotypes that did not meet the threshold to assign likely pathogenicity. Conclusion We define a novel developmental syndrome associated with biallelic hypomorphic variants in DHRS3; careful assessment of individual variants is required to establish a causal link to phenotype

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

p21-activated kinases are essential for spatial and temporal coordination of cytoskeletal dynamics with cellular adhesion during cell migration

Efeito inibitório em metaloproteinase de matriz

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas MonizAs metaloproteinases de matriz (MMPs) são um grupo de endopeptidases que contêm um ião de Zinco (Zn2+) no seu centro activo. Estas enzimas estão envolvidas em vários processos biológicos do organismo humano como por exemplo na embriogénese, na remodelação dos tecidos, na cicatrização e na angiogénese. A sua principal função é a degradação de proteínas da matriz extracelular controlando desta forma a extensão da remodelação da mesma. A actividade das MMPs é controlada por inibidores endógenos como a α2-macroglobulina e os inibidores tecidulares das metaloproteinases de matriz (TIMPs). Em condições normais existe um equilíbrio entre a actividade das MMPs e a actividade dos seus inibidores endógenos, no entanto, quando existe um desequilíbrio, o organismo deixa de ter a capacidade regular as MMPs e estas expressam em excesso a sua actividade o que pode provocar alguns processos patológicos, sendo os mais graves o cancro, a artrite e as doenças vasculares. O envolvimento destas enzimas nas referidas doenças serviu de impulsionador para despertar o interesse da comunidade científica, que nas últimas décadas tem tentado incessantemente desenvolver inibidores para poder controlar a actividade das MMPs e assim encontrar uma terapêutica para estas patologias. Apesar de terem sido desenvolvidos inibidores que comprovaram a efectividade da sua acção, estes acarretavam uma grande toxicidade que se traduzia numa serie de reacções adversas derivadas da sua instabilidade e falta de selectividade. Devido a tudo isto, nos últimos anos, aprofundou-se o estudo da estrutura química das MMPs e surgiu uma revolução no design de novos inibidores das metaloproteinases de matriz (MMPi) com o objectivo de sintetizar compostos que apresentem uma inibição mais potente aliada a especificidade necessária para que estes possam ser utilizados como terapêutica para algumas das mais graves doenças dos dias de hoje

NMR Studies of the C-Terminus of alpha4 Reveal Possible Mechanism of Its Interaction with MID1 and Protein Phosphatase 2A

Alpha4 is a regulatory subunit of the protein phosphatase family of enzymes and plays an essential role in regulating the catalytic subunit of PP2A (PP2Ac) within the rapamycin-sensitive signaling pathway. Alpha4 also interacts with MID1, a microtubule-associated ubiquitin E3 ligase that appears to regulate the function of PP2A. The C-terminal region of alpha4 plays a key role in the binding interaction of PP2Ac and MID1. Here we report on the solution structure of a 45-amino acid region derived from the C-terminus of alpha4 (alpha45) that binds tightly to MID1. In aqueous solution, alpha45 has properties of an intrinsically unstructured peptide although chemical shift index and dihedral angle estimation based on chemical shifts of backbone atoms indicate the presence of a transient α-helix. Alpha45 adopts a helix-turn-helix HEAT-like structure in 1% SDS micelles, which may mimic a negatively charged surface for which alpha45 could bind. Alpha45 binds tightly to the Bbox1 domain of MID1 in aqueous solution and adopts a structure consistent with the helix-turn-helix structure observed in 1% SDS. The structure of alpha45 reveals two distinct surfaces, one that can interact with a negatively charged surface, which is present on PP2A, and one that interacts with the Bbox1 domain of MID1