The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatinbinding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosomenucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.Key words: aneuploidy, centrosome hyperamplification, nuclear envelope architecture, spindle formation defects, Unc-84 The nuclear envelope (NE) separates the nuclear compartment from the cytoplasm. It is composed of two membranes: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The lumen between the two membranes is the perinuclear space (PNS). The ONM is continuous with the endoplasmic reticulum (ER), whereas the INM harbors a unique set of proteins. INM and ONM proteins can interact within the PNS. Underneath the INM, the nuclear lamina is located, which is formed by intermediate filament (IF) proteins and associated proteins. The lamina forms the nucleoskeleton and associates with the INM, chromatin and nuclear pore complexes. Proteins of the NE have important roles. They are involved in nuclear migration and positioning and are essential for many processes such as mitosis, meiosis, differentiation and cell migration. Furthermore, several of the NE proteins have been associated with inherited diseases (1,2). Research in mammalian cells and in Caenorhabditis eleganshas identified conserved components of the NE that link the nucleoskeleton to the cytoskeleton. In C. elegans, two putative INM proteins, matefin/SUN-1 and UNC-84, bind to the nuclear lamina and extend their C-terminus into the PNS where they interact with the C-termini of KASH domain proteins (Klarsicht/Anc-1/Syne homology, designated KASH domain). Matefin/SUN-1 and UNC-84 belong to the SUN family of proteins based on the presence of the conserved SUN (Sad1/UNC-84 homology) domain at their C-terminus. KASH domain proteins are type II transmembrane proteins of the NE and have been identified as molecular linkers connecting the nucleus to actin filaments [filamentous acti...

There is a well-defined regulatory framework governing the approval of chemicals for use as pharmaceuticals or release into the environment. Toxicity assessment is thus a major hurdle in the compound discovery pipeline, currently involving large scale animal testing. The search for alternative testing platforms is therefore an important priority. We have developed a convenient, low cost assay utilising the nematode Caenorhabditis elegans, to rapidly assess both acute toxicity and developmental and reproductive toxicity (DART). However the worm is protected by a robust cuticle that forms a barrier to chemical uptake. We assessed mutants with altered cuticle properties to identify sensitized strains optimized for toxicity assays. Evaluating the trade-off between increased permeability and reduced fitness identifies bus-5(br19) as the most suitable strain for chemical exposure. We demonstrate the applicability of this assay for a range of chemicals with differing properties, including a modified exposure protocol for volatile or less soluble compounds. This work enhances the effectiveness of C. elegans for convenient toxicity assessment, which could contribute to a reduction in the use of vertebrates particularly at the crucial early stages of product development. Strains identified in this work will also enhance the sensitivity of C. elegans based drug discovery platforms.

Regulated movements of the nucleus are essential during zygote formation, cell migrations, and differentiation of neurons. The nucleus moves along microtubules (MTs) and is repositioned on F-actin at the cellular cortex. Two families of nuclear envelope proteins, SUN and KASH, link the nucleus to the actin and MT cytoskeletons during nuclear movements. However, the role of actin nucleators in nuclear migration and positioning is poorly understood. We show that the branched actin nucleator, Arp2/3, affects nuclear movements throughout embryonic and larval development in C. elegans, including nuclear migrations in epidermal cells and neuronal precursors. In one-cell embryos the migration of the male pronucleus to meet the female pronucleus after fertilization requires Arp2/3. Loss of Arp2/3 or its activators changes the dynamics of non-muscle myosin, NMY-2, and alters the cortical accumulation of posterior PAR proteins. Reduced establishment of the posterior microtubule cytoskeleton in Arp2/3 mutants correlates with reduced male pronuclear migration. The UNC-84/SUN nuclear envelope protein that links the nucleus to the MT and actin cytoskeleton is known to regulate later nuclear migrations. We show here it also positions the male pronucleus. These studies demonstrate a global role for Arp2/3 in nuclear migrations. In the C. elegans one-cell embryo Arp2/3 promotes the establishment of anterior/posterior polarity and promotes MT growth that propels the anterior migration of the male pronucleus. In contrast with previous studies emphasizing pulling forces on the male pronucleus, we propose that robust MT nucleation pushes the male pronucleus anteriorly to join the female pronucleus.

The Dictyostelium centrosome is a nucleus associated body consisting of a box-shaped core surrounded by the corona, an amorphous matrix functionally equivalent to the pericentriolar material of animal centrosomes which is responsible for the nucleation and anchoring of microtubules. Here we describe CP250 a component of the corona, an acidic coiled coil protein that is present at the centrosome throughout interphase while disappearing during prophase and reappearing at the end of late telophase. Amino acids 756-1148 of the 2110 amino acids are sufficient for centrosomal targeting and cell cycle-dependent centrosome association. Mutant cells lacking CP250 are smaller in size, growth on bacteria is delayed, chemotaxis is altered, and development is affected, which, in general, are defects observed in cytoskeletal mutants. Furthermore, loss of CP250 affected the nuclear envelope and led to reduced amounts and altered distribution of Sun-1, a conserved nuclear envelope protein that connects the centrosome to chromatin.

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Platelets undergo profound shape changes upon adhesion to damaged blood vessel walls that are mediated by reorganisation of the actin cytoskeleton in response to receptor-mediated signalling cascades. The highly conserved 56 kDa multidomain cyclase associated protein 1 (CAP1) works in concert with cofilin and profilin to modulate actin filament turnover by facilitating cofilin-mediated actin filament severing and depolymerisation and catalysing profilin-mediated regeneration of actin monomers for reutilisation in growing filaments. CAP1 is abundant in platelets but its roles remain unexplored. We report that in suspended platelets CAP1 localises predominantly at the cell cortex whereas in spread platelets it is uniformly distributed in the cytoplasm, with enrichment at the cell cortex and the periphery of actin nodules. Upon subcellular fractionation most CAP1 was found cytosolic but part associated to the membrane fraction in an actin-independent manner. Interestingly, upon stimulation with thrombin a significant proportion of the membrane-associated CAP1 translocates to the cytosol. This relocalisation was prevented by prior treatment with PGI2 or the nitric oxide donor GSNO, or by inhibition of GSK3. Our results place CAP1 at a crossroad of signalling pathways that control platelet activation by contributing to actin remodelling at the cell cortex and actin nodules during platelet spreading.

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum makes use of Rho-regulated signaling pathways to reorganize its cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation competent cells with the chemoattractant cAMP.

The model organism D. discoideum is well suited to investigate basic questions of molecular and cell biology, particularly those related to the structure, regulation, and dynamics of the cytoskeleton, signal transduction, cell-cell adhesion, and development. D. discoideum cells make use of Rho-regulated signaling pathways to reorganize the actin cytoskeleton during chemotaxis, endocytosis, and cytokinesis. In this organism the Rho family encompasses 20 members, several belonging to the Rac subfamily, but there are no representatives of the Cdc42 and Rho subfamilies. Here we present protocols suitable for monitoring the actin polymerization response and the activation of Rac upon stimulation of aggregation-competent cells with the chemoattractant cAMP, and for monitoring the localization and dynamics of Rac activity in live cells.