With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.

The outer nuclear membrane proteins nesprin-1 and nesprin-2 are retained at the nuclear envelope through an interaction of their klarsicht/ANC-1/syne homology (KASH) domain with Sun proteins present at the inner nuclear membrane. We investigated the requirements for the localization of nesprin-3α at the outer nuclear membrane and show that the mechanism by which its localization is mediated is similar to that reported for the localization of nesprin-1 and nesprin-2: the last four amino acids of the nesprin-3α KASH domain are essential for its interaction with Sun1 and Sun2. Moreover, deletion of these amino acids or knockdown of the Sun proteins results in a redistribution of nesprin-3α away from the nuclear envelope and into the endoplasmic reticulum (ER), where it becomes colocalized with the cytoskeletal crosslinker protein plectin. Both nesprin-3α and plectin can form dimers, and dimerization of plectin is required for its interaction with nesprin-3α at the nuclear envelope, which is mediated by its N-terminal actin-binding domain. Additionally, overexpression of the plectin actin-binding domain stabilizes the actin cytoskeleton and prevents the recruitment of endogenous plectin to the nuclear envelope. Our studies support a model in which the actin cytoskeleton influences the binding of plectin dimers to dimers of nesprin-3α, which in turn are retained at the nuclear envelope through an interaction with Sun proteins.

The nucleus in eukaryotic cells can move within the cytoplasm, and its position is crucial for many cellular events, including migration and differentiation. Nuclear anchorage and movement can be achieved through association of outer nuclear membrane (ONM) proteins with the three cytoskeletal systems. Two decades ago studies described C. elegans mutants with defects in such events, but only recently has it been shown that the strategies for nuclear positioning are indeed conserved in C. elegans, Drosophila, mammals and potentially all eukaryotes. The integral ONM proteins implicated in these processes thus far all contain a conserved Klarsicht/ANC-1/Syne homology (KASH) domain at their C-terminus that can associate with Sad1p/UNC-84 (SUN)-domain proteins of the inner nuclear membrane within the periplasmic space of the nuclear envelope (NE). The complex thus formed is responsible not only for association with cytoplasmic elements but also for the integrity of the NE itself. Journal of Cell Science 5022 region and residues that lie within the PS, and a cytoplasmic N-terminus that does not show sequence similarity to any known protein (McGee et al., 2006). UNC-84 contains several potential transmembrane domains and a C-terminal region that is homologous to the yeast protein Sad1p and, accordingly, is called the Sad1p/UNC-84 (SUN) domain (Malone et al., 1999;McGee et al., 2006). The localization of UNC-84 to the INM is not dependent upon its SUN domain but rather on the presence of nuclear lamins, which probably interact with its Nterminus (Lee et al., 2002). Deletion of UNC-84, mutations in the UNC-84 SUN domain or mutations within the UNC-83 KASH domain can prevent the localization of UNC-83 at the ONM (McGee et al., 2006;Starr et al., 2001), presumably because of a loss of direct interaction between the UNC-83 KASH and UNC-84 SUN domains within the PS (McGee et al., 2006) (Fig. 2). This explains why the nuclear migration defects in unc-83 and unc-84 mutant worms are very similar (Malone et al., 1999). Curiously, UNC-83 is present at the NE in a limited number of cell types (including P, hyp7, intestinal, pharyngeal and uterine cells), unlike UNC-84, which is localized at the NE in nearly all cells (Starr et al., 2001).UNC-83 and UNC-84 were originally proposed to tether the nucleus to centrosomes, which was hypothesized to drive nuclear migration (Malone et al., 1999;Reinsch and Gonczy, 1998); however, later studies showed a normal association between the centrosomes and nuclei in unc-83 and unc-84 mutant cells whose nuclei fail to migrate (Lee et al., 2002;Starr et al., 2001). The proteins that interact with the N-terminus of UNC-83 to facilitate nuclear migration have not been identified. Recently, another ONM protein, was shown to mediate an association between the centrosomes and Journal of Cell Science 119 (24) the nucleus in C. elegans. In zygote defective (zyg)-12 mutant worms, the centrosome detachment defect in the developing embryo results in death as a consequence of chromosome segregation defects (...

Nesprin-3 regulates perinuclear localization of plectin and vimentin in Sertoli cells but is dispensable for Sertoli cell function in spermatogenesis. In addition, nuclear positioning and anchorage are not disturbed in nesprin-3–knockout mice.

SummaryThe outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3a and nesprin-3b. Nesprin-3b is only expressed during early development and lacks seven amino acids in its first spectrin repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin b4 subunit are also important for the binding to nesprin-3a, indicating partial overlapping binding sequences for nesprin-3a and integrin b4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.

The cytoskeleton is connected to the nuclear interior by LINC (linker of nucleoskeleton and cytoskeleton) complexes located in the nuclear envelope. These complexes consist of SUN proteins and nesprins present in the inner and outer nuclear membrane respectively. Whereas SUN proteins can bind the nuclear lamina, members of the nesprin protein family connect the nucleus to different components of the cytoskeleton. Nesprin-1 and -2 can establish a direct link with actin filaments, whereas nesprin-4 associates indirectly with microtubules through its interaction with kinesin-1. Nesprin-3 is the only family member known that can link the nuclear envelope to intermediate filaments. This indirect interaction is mediated by the binding of nesprin-3 to the cytoskeletal linker protein plectin. Furthermore, nesprin-3 can connect the nucleus to microtubules by its interactions with BPAG1 (bullous pemphigoid antigen 1) and MACF (microtubule-actin cross-linking factor). In contrast with the active roles that nesprin-1, -2 and -4 have in actin- and microtubule-dependent nuclear positioning, the role of nesprin-3 is likely to be more passive. We suggest that it helps to stabilize the anchorage of the nucleus within the cytoplasm and maintain the structural integrity and shape of the nucleus.

Plectin is a cytoskeletal linker protein that consists of a central rod domain connecting two globular domains. Rodless plectin is able to functionally compensate for the loss of full-length plectin in mice and, like full-length plectin, is able to form dimers.