Jan Gettemans

Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy

Charcot-Marie-Tooth disease (CMT) is the most common inherited neuromuscular disease and is characterized by considerable clinical and genetic heterogeneity. We previously reported a Russian family with autosomal dominant axonal CMT and assigned the locus underlying the disease (CMT2F; OMIM 606595) to chromosome 7q11–q21 (ref. 2). Here we report a missense mutation in the gene encoding 27-kDa small heat-shock protein B1 (HSPB1, also called HSP27) that segregates in the family with CMT2F. Screening for mutations in HSPB1 in 301 individuals with CMT and 115 individuals with distal hereditary motor neuropathies (distal HMNs) confirmed the previously observed mutation and identified four additional missense mutations. We observed the additional HSPB1 mutations in four families with distal HMN and in one individual with CMT neuropathy. Four mutations are located in the Hsp20–α-crystallin domain, and one mutation is in the C-terminal part of the HSP27 protein. Neuronal cells transfected with mutated HSPB1 were less viable than cells expressing the wild-type protein. Cotransfection of neurofilament light chain (NEFL) and mutant HSPB1 resulted in altered neurofilament assembly in cells devoid of cytoplasmic intermediate filaments.

Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy

Distal hereditary motor neuropathies are pure motor disorders of the peripheral nervous system resulting in severe atrophy and wasting of distal limb muscles. In two pedigrees with distal hereditary motor neuropathy type II linked to chromosome 12q24.3, we identified the same mutation (K141N) in small heat-shock 22-kDa protein 8 (encoded by HSPB8; also called HSP22). We found a second mutation (K141E) in two smaller families. Both mutations target the same amino acid, which is essential to the structural and functional integrity of the small heat-shock protein αA-crystallin. This positively charged residue, when mutated in other small heat-shock proteins, results in various human disorders. Coimmunoprecipitation experiments showed greater binding of both HSPB8 mutants to the interacting partner HSPB1. Expression of mutant HSPB8 in cultured cells promoted formation of intracellular aggregates. Our findings provide further evidence that mutations in heat-shock proteins have an important role in neurodegenerative disorders.

Disrupted function and axonal distribution of mutant tyrosyl-tRNA synthetase in dominant intermediate Charcot-Marie-Tooth neuropathy

Charcot-Marie-Tooth (CMT) neuropathies are common disorders of the peripheral nervous system caused by demyelination or axonal degeneration, or a combination of both features. We previously assigned the locus for autosomal dominant intermediate CMT neuropathy type C (DI-CMTC) to chromosome 1p34-p35. Here we identify two heterozygous missense mutations (G41R and E196K) and one de novo deletion (153–156delVKQV) in tyrosyl-tRNA synthetase (YARS) in three unrelated families affected with DI-CMTC. Biochemical experiments and genetic complementation in yeast show partial loss of aminoacylation activity of the mutant proteins, and mutations in YARS, or in its yeast ortholog TYS1, reduce yeast growth. YARS localizes to axonal termini in differentiating primary motor neuron and neuroblastoma cultures. This specific distribution is significantly reduced in cells expressing mutant YARS proteins. YARS is the second aminoacyl-tRNA synthetase found to be involved in CMT, thereby linking protein-synthesizing complexes with neurodegeneration.

YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3ζ

The Golgi apparatus has long been suggested to be important for directing secretion to specific sites on the plasma membrane in response to extracellular signaling events. However, the mechanisms by which signaling events are coordinated with Golgi apparatus function remain poorly understood. Here, we identify a scaffolding function for the Golgi matrix protein GM130 that sheds light on how such signaling events may be regulated. We show that the mammalian Ste20 kinases YSK1 and MST4 target to the Golgi apparatus via the Golgi matrix protein GM130. In addition, GM130 binding activates these kinases by promoting autophosphorylation of a conserved threonine within the T-loop. Interference with YSK1 function perturbs perinuclear Golgi organization, cell migration, and invasion into type I collagen. A biochemical screen identifies 14-3-3ζ as a specific substrate for YSK1 that localizes to the Golgi apparatus, and potentially links YSK1 signaling at the Golgi apparatus with protein transport events, cell adhesion, and polarity complexes important for cell migration.

PIP2-PDZ Domain Binding Controls the Association of Syntenin with the Plasma Membrane

PDZ proteins organize multiprotein signaling complexes. According to current views, PDZ domains engage in protein-protein interactions. Here we show that the PDZ domains of several proteins bind phosphatidylinositol 4,5-bisphosphate (PIP(2)). High-affinity binding of syntenin to PIP(2)-containing lipid layers requires both PDZ domains of this protein. Competition and mutagenesis experiments reveal that the protein and the PIP(2) binding sites in the PDZ domains overlap. Overlay assays indicate that the two PDZ domains of syntenin cooperate in binding to cognate peptides and PIP(2). Experiments on living cells demonstrate PIP(2)-dependent and peptide-dependent modes of plasma membrane association of the PDZ domains of syntenin. These observations suggest that local changes in phosphoinositide concentration control the association of PDZ proteins with their target receptors at the plasma membrane.

Functional complexes between YAP2 and ZO-2 are PDZ domain-dependent, and regulate YAP2 nuclear localization and signalling.

The Hippo pathway regulates the size of organs by controlling two opposing processes: proliferation and apoptosis. YAP2 (Yes kinase-associated protein 2), one of the three isoforms of YAP, is a WW domain-containing transcriptional co-activator that acts as the effector of the Hippo pathway in mammalian cells. In addition to WW domains, YAP2 has a PDZ-binding motif at its C-terminus. We reported previously that this motif was necessary for YAP2 localization in the nucleus and for promoting cell detachment and apoptosis. In the present study, we show that the tight junction protein ZO (zonula occludens)-2 uses its first PDZ domain to form a complex with YAP2. The endogenous ZO-2 and YAP2 proteins co-localize in the nucleus. We also found that ZO-2 facilitates the nuclear localization and pro-apoptotic function of YAP2, and that this activity of ZO-2 is PDZ-domain-dependent. The present paper is the first report on a PDZ-based nuclear translocation mechanism. Moreover, since the Hippo pathway acts as a tumour suppressor pathway, the YAP2-ZO-2 complex could represent a target for cancer therapy.

Plastins: versatile modulators of actin organization in (patho)physiological cellular processes

AbstractMany actin-binding proteins are expressed in eukaryotic cells. These polypep-tides assist in stabilizing and rearranging the organization of the actin cytoskeleton in response to external stimuli, or during cell migration and adhesion. Here we review a particular set of actin-binding proteins called plastins. Plastins (also called fimbrins) belong to a subclass of actin-binding proteins known as actin bundling proteins. Three isoforms have been characterized in mammals: T-plastin is expressed in cells from solid tissue, whereas L-plastin occurs predominantly in hematopoietic cells. The third isoform, I-plastin, is specifically expressed in the small intestine, colon and kidney. These proteins share the unique property of cross-linking actin filaments into tight bundles. Although plastins are primarily involved in regulation of the actin cytoskeleton, they possess some unique features. For instance, they are implicated in invasion by pathogenic bacteria such as Shigella flexneri and Salmonella typhimurium. Also, L-plastin plays an important role in leukocyte function. T-plastin, on the other hand, is possibly involved in DNA repair. Finally, both T- and L-plastin are implicated in several diseases, and L-plastin is considered to be a valuable marker for cancer.

Gelsolin-induced epithelial cell invasion is dependent on Ras-Rac signaling.

Gelsolin is a widely distributed actin binding protein involved in controlling cell morphology, motility, signaling and apoptosis. The role of gelsolin in tumor progression, however, remains poorly understood. Here we show that expression of green fluorescent pro tein (GFP)‐tagged gelsolin in MDCK‐AZ, MDCKtsSrc or HEK293T cells promotes invasion into collagen type I. In organ culture assays, MDCK cells expressing gelsolin–GFP invaded pre‐cultured chick heart fragments. Gelsolin expression inhibited E‐cadherin‐mediated cell aggregation but did not disrupt the E‐cadherin–catenin complex. Co‐expression of dominant‐negative Rac1N17, but not RhoAN19 or Cdc42N17, counteracted gelsolin‐induced invasion, suggesting a requirement for Rac1 activity. Increased ARF6, PLD or PIP5K 1α activity canceled out gelsolin‐induced invasion. Furthermore, we found that invasion induced by gelsolin is dependent on Ras activity, acting through the PI3K–Rac pathway via the Ras guanine nucleotide exchange factor Sos‐1. These findings establish a connection between gelsolin and the Ras oncogenic signaling pathway.

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid.

An extensive survey was carried out for compounds capable of regulating actin‐binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5‐P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP–actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine‐1‐phosphate were inactive. Furthermore, LPA inhibited the F‐actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin‐containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5‐P2. We further show that binding of LPA to these actin‐regulatory proteins promotes their phosphorylation by pp60c‐src. A PI 4,5‐P2‐binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin‐binding proteins. When both LPA and PI 4,5‐P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5‐P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside‐permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin‐binding proteins. Our findings can also explain agonist‐induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.

EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research

We argue that the field of extracellular vesicle (EV) biology needs more transparent reporting to facilitate interpretation and replication of experiments. To achieve this, we describe EV-TRACK, a crowdsourcing knowledgebase (http://evtrack.org) that centralizes EV biology and methodology with the goal of stimulating authors, reviewers, editors and funders to put experimental guidelines into practice.