Kees Straatman

Muscular dystrophy-associated SUN1 and SUN2 variants disrupt nuclear-cytoskeletal connections and myonuclear organization.

Proteins of the nuclear envelope (NE) are associated with a range of inherited disorders, most commonly involving muscular dystrophy and cardiomyopathy, as exemplified by Emery-Dreifuss muscular dystrophy (EDMD). EDMD is both genetically and phenotypically variable, and some evidence of modifier genes has been reported. Six genes have so far been linked to EDMD, four encoding proteins associated with the LINC complex that connects the nucleus to the cytoskeleton. However, 50% of patients have no identifiable mutations in these genes. Using a candidate approach, we have identified putative disease-causing variants in the SUN1 and SUN2 genes, also encoding LINC complex components, in patients with EDMD and related myopathies. Our data also suggest that SUN1 and SUN2 can act as disease modifier genes in individuals with co-segregating mutations in other EDMD genes. Five SUN1/SUN2 variants examined impaired rearward nuclear repositioning in fibroblasts, confirming defective LINC complex function in nuclear-cytoskeletal coupling. Furthermore, myotubes from a patient carrying compound heterozygous SUN1 mutations displayed gross defects in myonuclear organization. This was accompanied by loss of recruitment of centrosomal marker, pericentrin, to the NE and impaired microtubule nucleation at the NE, events that are required for correct myonuclear arrangement. These defects were recapitulated in C2C12 myotubes expressing exogenous SUN1 variants, demonstrating a direct link between SUN1 mutation and impairment of nuclear-microtubule coupling and myonuclear positioning. Our findings strongly support an important role for SUN1 and SUN2 in muscle disease pathogenesis and support the hypothesis that defects in the LINC complex contribute to disease pathology through disruption of nuclear-microtubule association, resulting in defective myonuclear positioning.

Molecular Dissection of the Centrosome Overduplication Pathway in S-Phase-Arrested Cells

ABSTRACT Cancer cells frequently exhibit overduplicated centrosomes that lead to formation of multipolar spindles, chromosome missegregation, and aneuploidy. However, the molecular events involved in centrosome overduplication remain largely unknown. Experimentally, centrosome overduplication is observed in p53-deficient cells arrested in S phase with hydroxyurea. Using this assay, we have identified distinct roles for Cdk2, microtubules, dynein, and Hsp90 in the overduplication of functional centrosomes in mammalian cells and show that Cdk2 is also required for the generation of centriolar satellites. Moreover, we demonstrate that nuclear export is required for centriolar satellite formation and centrosome overduplication, with export inhibitors causing a Cdk-dependent accumulation of nuclear centrin granules. Hence, we propose that centrosome precursors may arise in the nucleus, providing a novel mechanistic explanation for how nuclear Cdk2 can promote centrosome overduplication in the cytoplasm. Furthermore, this study defines a molecular pathway that may be targeted to prevent centrosome overduplication in S-phase-arrested cancer cells.

DJ-1 interactions with α -synuclein attenuate aggregation and cellular toxicity in models of Parkinson’s disease

Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized by the loss of neurons in the substantia nigra pars compacta and the presence of Lewy bodies in surviving neurons. These intracellular protein inclusions are primarily composed of misfolded α-synuclein (aSyn), which has also been genetically linked to familial and sporadic forms of PD. DJ-1 is a small ubiquitously expressed protein implicated in several pathways associated with PD pathogenesis. Although mutations in the gene encoding DJ-1 lead to familial early-onset PD, the exact mechanisms responsible for its role in PD pathogenesis are still elusive. Previous work has found that DJ-1 – which has protein chaperone-like activity – modulates aSyn aggregation. Here, we investigated possible physical interactions between aSyn and DJ-1 and any consequent functional and pathological relevance. We found that DJ-1 interacts directly with aSyn monomers and oligomers in vitro, and that this also occurs in living cells. Notably, several PD-causing mutations in DJ-1 constrain this interaction. In addition, we found that overexpression of DJ-1 reduces aSyn dimerization, whereas mutant forms of DJ-1 impair this process. Finally, we found that human DJ-1 as well as yeast orthologs of DJ-1 reversed aSyn-dependent cellular toxicity in Saccharomyces cerevisiae. Taken together, these data suggest that direct interactions between DJ-1 and aSyn constitute the basis for a neuroprotective mechanism and that familial mutations in DJ-1 may contribute to PD by disrupting these interactions.

MicroRNA expression profiling in patients with lamin A/C-associated muscular dystrophy

Mutations in the lamin A/C gene (LMNA) cause several disorders referred to as laminopathies, which include premature aging syndromes, lipodystrophy, and striated muscle disorders. There is evidence that lamin A/C plays a role in gene expression. MicroRNAs (miRNAs) are short noncoding RNAs regulating mRNAs involved in various biological processes, including the pathophysiology of striated muscles. Here, we profiled the expression of the miRNA transcriptome in skeletal muscle from patients with LMNA‐related muscular dystrophy. Results show that control and patient groups can be distinguished based on their miRNA expression profile. Sixteen miRNAs are significantly dysregulated in patients compared with controls. Pathway enrichment analysis in the predicted targets of these miRNAs revealed pathways involved in muscle repair, such as MAPK, transforming growth factor‐β, and Wnt signaling. Interestingly, 9 of these miRNAs (hsa‐miR‐100, −127–3p, −148a, −136∗, −192, −335, −376c, −489, and −502–3p) are highly expressed in fetal muscle, suggesting that the fetal miRNA gene program mediates a regenerative process. Overexpression of these miRNAs in C2C12 mouse myoblasts revealed that 3 of them (miR‐100, −192, and −335) participate in muscle proliferation and differentiation. We identified target genes that likely mediate this effect, which include the calcineurin gene PPP3CA. Our findings are the first to demonstrate that miRNA expression is affected in laminopathies.—Sylvius, N., Bonne, G., Straatman, K., Reddy, T., Gant, T. W., Shackleton, S. MicroRNA expression profiling in patients with lamin A/C‐associated muscular dystrophy. FASEB J. 25, 3966–3978 (2011). www.fasebj.org

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

Poc1A and Poc1B act together in human cells to ensure centriole integrity

Summary Proteomic studies in unicellular eukaryotes identified a set of centriolar proteins that included proteome of centriole 1 (Poc1). Functional studies in these organisms implicated Poc1 in centriole duplication and length control, as well as ciliogenesis. Using isoform-specific antibodies and RNAi depletion, we have examined the function of the two related human proteins, Poc1A and Poc1B. We find that Poc1A and Poc1B each localize to centrioles and spindle poles, but do so independently and with different dynamics. However, although loss of one or other Poc1 protein does not obviously disrupt mitosis, depletion of both proteins leads to defects in spindle organization with the generation of unequal or monopolar spindles. Our data indicate that, once incorporated, a fraction of Poc1A and Poc1B remains stably associated with parental centrioles, but that depletion prevents incorporation into nascent centrioles. Nascent centrioles lacking both Poc1A and Poc1B exhibit loss of integrity and maturation, and fail to undergo duplication. Thus, when Poc1A and Poc1B are co-depleted, new centrosomes capable of maturation cannot assemble and unequal spindles result. Interestingly, Poc1B, but not Poc1A, is phosphorylated in mitosis, and depletion of Poc1B alone was sufficient to perturb cell proliferation. Hence, Poc1A and Poc1B play redundant, but essential, roles in generation of stable centrioles, but Poc1B may have additional independent functions during cell cycle progression.

Abdominal fat distribution and its relationship to brain changes: the differential effects of age on cerebellar structure and function: a cross-sectional, exploratory study

Objectives To investigate whether the metabolically important visceral adipose tissue (VAT) relates differently to structural and functional brain changes in comparison with body weight measured as body mass index (BMI). Moreover, we aimed to investigate whether these effects change with age. Design Cross-sectional, exploratory. Setting University Clinic, Integrative Research and Treatment Centre. Participants We included 100 (mean BMI=26.0 kg/m², 42 women) out of 202 volunteers randomly invited by the citys registration office, subdivided into two age groups: young-to-mid-age (n=51, 20–45 years of age, mean BMI=24.9, 24 women) versus old (n=49, 65–70 years of age, mean BMI=27.0, 18 women). Main outcome measures VAT, BMI, subcutaneous abdominal adipose tissue, brain structure (grey matter density), functional brain architecture (eigenvector centrality, EC). Results We discovered a loss of cerebellar structure with increasing VAT in the younger participants, most significantly in regions involved in motor processing. This negative correlation disappeared in the elderly. Investigating functional brain architecture showed again inverse VAT–cerebellum correlations, whereas now regions involved in cognitive and emotional processing were significant. Although we detected similar results for EC using BMI, significant age interaction for both brain structure and functional architecture was only found using VAT. Conclusions Visceral adiposity is associated with cerebellar changes of both structure and function, whereas the regions involved contribute to motor, cognitive and emotional processes. Furthermore, these associations seem to be age dependent, with younger adults’ brains being adversely affected.

CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells.

CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion.

Is high-resolution spectral domain optical coherence tomography reliable in nystagmus?

Recently, there has been an increased number of studies investigating retinal morphology in infantile nystagmus.1–4 Optical coherence tomography (OCT) studies in achromatopsia have shown progressive retinal changes.1 2 4 Most OCT software limits segmentation to the retinal nerve fibre layer and overall retinal thickness (RT). However, we have shown that outer nuclear layer (ONL), outer segment (OS) and foveal depth (FD) are important measurements in infantile nystagmus.2 5 Intraretinal thicknesses (FD, ONL, inner segment (IS) and OS) can be derived using reflectivity-based segmentation using ImageJ1 (figure 1). External limiting membrane (ELM) and inner–outer segment junction (IS/OS) reflectivity changes have been reported in blue cone monochromatism and achromatopsia. However, it is unclear whether nystagmus allows reproducible OCT measurements …

Fluorescent labelling of nascent RNA reveals nuclear transcription domains throughout plant cell nuclei

SummaryTranscription and RNA processing are main functions of the nucleus. These processes are found localized in specific nuclear domains. We have investigated the presence of nuclear transcription domains in plants by incorporating 5-bromouridine 5′-triphosphate in nascent RNA in isolated nuclei ofBrassica napus. Bromo-uridine labelled RNA was visualized by a FITC-labelled biotin-avidin system in combination with confocal laser scanning microscopy. Labelled domains were found throughout the nucleus, in some cases including the nucleolus. This shows that the distribution of transcription sites in plant nuclei is similar to that of mammalian nuclei and that the same labelling procedures can be used.