Localization and function of calmodulin in live-cells of Aspergillus nidulans

Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca²⁺. Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP–CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP–CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP–CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.     

A Novel Role for the Centrosomal Protein,Pericentrin, in Regulation of Insulin Secretory Vesicle Docking in Mouse Pancreatic β-cells

The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic β-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in β-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in β-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory β-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.     

Calmodulin concentrates at the apex of growing hyphae and localizes to the Spitzenkörper in Aspergillus nidulans

The calmodulin (CaM) localization pattern in the growing hyphal tip of Aspergillus nidulans was studied with the functional GFP::CaM fusion protein. A faint tip-high gradient of CaM was found in the growing hyphal tip, with CaM highly localized in the region corresponding to the Spitzenk?rper forming a bright granule. The position of highly concentrated CaM in the extreme apex seemed to determine the orientation of the hypha. The normal pattern of CaM localization was also shown to be dependent on the integrated actin cytoskeleton. When the growth of the hyphal tip ceased, CaM failed to localize in the bright granule and was evenly distributed in the hyphal tip. These findings suggest that CaM may play an important role in establishing and maintaining apical organization, morphogenesis, and growth in Aspergillus nidulans.     

Mitosis-specific anchoring of gamma tubulin complexes by pericentrin controls spindle organization and mitotic entry

Microtubule nucleation is the best known function of centrosomes. Centrosomal microtubule nucleation is mediated primarily by gamma tubulin ring complexes (gamma TuRCs). However, little is known about the molecules that anchor these complexes to centrosomes. In this study, we show that the centrosomal coiled-coil protein pericentrin anchors gamma TuRCs at spindle poles through an interaction with gamma tubulin complex proteins 2 and 3 (GCP2/3). Pericentrin silencing by small interfering RNAs in somatic cells disrupted gamma tubulin localization and spindle organization in mitosis but had no effect on gamma tubulin localization or microtubule organization in interphase cells. Similarly, overexpression of the GCP2/3 binding domain of pericentrin disrupted the endogenous pericentrin-gamma TuRC interaction and perturbed astral microtubules and spindle bipolarity. When added to Xenopus mitotic extracts, this domain uncoupled gamma TuRCs from centrosomes, inhibited microtubule aster assembly, and induced rapid disassembly of preassembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding and were specific for mitotic centrosomal asters as we observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Additionally, pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. We conclude that pericentrin anchoring of gamma tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death.     

Pericentrin and γ-Tubulin Form a Protein Complex and Are Organized into a Novel Lattice at the Centrosome

Pericentrin and γ-tubulin are integral centrosome proteins that play a role in microtubule nucleation and organization. In this study, we examined the relationship between these proteins in the cytoplasm and at the centrosome. In extracts prepared from Xenopus eggs, the proteins were part of a large complex as demonstrated by sucrose gradient sedimentation, gel filtration and coimmunoprecipitation analysis. The pericentrin–γ-tubulin complex was distinct from the previously described γ-tubulin ring complex (γ-TuRC) as purified γ-TuRC fractions did not contain detectable pericentrin. When assembled at the centrosome, the two proteins remained in close proximity as shown by fluorescence resonance energy transfer. The three- dimensional organization of the centrosome-associated fraction of these proteins was determined using an improved immunofluorescence method. This analysis revealed a novel reticular lattice that was conserved from mammals to amphibians, and was organized independent of centrioles. The lattice changed dramatically during the cell cycle, enlarging from G1 until mitosis, then rapidly disassembling as cells exited mitosis. In cells colabeled to detect centrosomes and nucleated microtubules, lattice elements appeared to contact the minus ends of nucleated microtubules. Our results indicate that pericentrin and γ-tubulin assemble into a unique centrosome lattice that represents the higher-order organization of microtubule nucleating sites at the centrosome.     

Ras1-induced hyphal development in Candida albicans requires the formin Bni1

Formins are downstream effector proteins of Rho-type GTPases and are involved in the organization of the actin cytoskeleton and actin cable assembly at sites of polarized cell growth. Here we show using in vivo time-lapse microscopy that deletion of the Candida albicans formin homolog BNI1 results in polarity defects during yeast growth and hyphal stages. Deletion of the second C. albicans formin, BNR1, resulted in elongated yeast cells with cell separation defects but did not interfere with the ability of bnr1 cells to initiate and maintain polarized hyphal growth. Yeast bni1 cells were swollen, showed an increased random budding pattern, and had a severe defect in cytokinesis, with enlarged bud necks. Induction of hyphal development in bni1 cells resulted in germ tube formation but was halted at the step of polarity maintenance. Bni1-green fluorescent protein is found persistently at the hyphal tip and colocalizes with a structure resembling the Spitzenk?rper of true filamentous fungi. Introduction of constitutively active ras1G13V in the bni1 strain or addition of cyclic AMP to the growth medium did not bypass bni1 hyphal growth defects. Similarly, these agents were not able to suppress hyphal growth defects in the wal1 mutant which is lacking the Wiskott-Aldrich syndrome protein (WASP) homolog. These results suggest that the maintenance of polarized hyphal growth in C. albicans requires coordinated regulation of two actin cytoskeletal pathways, including formin-mediated secretion and WASP-dependent endocytosis.     

Direct interaction of pericentrin with cytoplasmic dynein light intermediate chain contributes to mitotic spindle organization.

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150(Glued) subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin-dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.     

Traffic of chitin synthase 1 (CHS-1) to the Spitzenkörper and developing septa in hyphae of Neurospora crassa: actin dependence and evidence of distinct microvesicle populations

We describe the subcellular location of chitin synthase 1 (CHS-1), one of seven chitin synthases in Neurospora crassa. Laser scanning confocal microscopy of growing hyphae showed CHS-1–green fluorescent protein (GFP) localized conspicuously in regions of active wall synthesis, namely, the core of the Spitzenkörper (Spk), the apical cell surface, and developing septa. It was also present in numerous fine particles throughout the cytoplasm plus some large vacuoles in distal hyphal regions. Although the same general subcellular distribution was observed previously for CHS-3 and CHS-6, they did not fully colocalize. Dual labeling showed that the three different chitin synthases were contained in different vesicular compartments, suggesting the existence of a different subpopulation of chitosomes for each CHS. CHS-1–GFP persisted in the Spk during hyphal elongation but disappeared from the septum after its development was completed. Wide-field fluorescence microscopy and total internal reflection fluorescence microscopy revealed subapical clouds of particles, suggestive of chitosomes moving continuously toward the Spk. Benomyl had no effect on CHS-1–GFP localization, indicating that microtubules are not strictly required for CHS trafficking to the hyphal apex. Conversely, actin inhibitors caused severe mislocalization of CHS-1–GFP, indicating that actin plays a major role in the orderly traffic and localization of CHS-1 at the apex.     

The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin

AKAP450 (also known as AKAP350, CG-NAP or Hyperion) and pericentrin are large coiled-coil proteins found in mammalian centrosomes that serve to recruit structural and regulatory components including dynein and protein kinase A. We find that these proteins share a well conserved 90 amino acid domain near their C-termini that is also found in coiled-coil proteins of unknown function from Drosophila and fission yeast. Fusion of the C-terminal region from either protein to a reporter protein confers a centrosomal localization, and overexpression of the domain from AKAP450 displaces endogenous pericentrin, suggesting recruitment to a shared site. When isolated from transfected cells the C-terminal domain of AKAP450 was associated with calmodulin, suggesting that this protein could contribute to centrosome assembly.     

Aberrant microtubule organization in dividing root cells of p60-katanin mutants

Aberrant microtubule organization has been recently recorded in dividing root cells of fra2 and lue1 p60-katanin Arabidopsis thaliana mutants. Here, we report similar defects in the bot1 and ktn1-2 mutants of the same plant, proposing that they constitute a consistent phenotype of p60-katanin mutants. In addition, we show that the Targeting Protein for Xklp2 (TPX2) protein co-localizes with microtubules on the surface of prophase nuclei of the mutants, probably participating in multipolar spindle assembly. As microtubule organization defects are not observed in metaphase/anaphase spindles and initiating phragmoplasts, we also discuss the putative association of the observed aberrations with the nuclear envelope and we emphasize on the mechanism of bipolar metaphase spindle organization in the mutants. It seems that chromosome-mediated spindle assembly, probably minimally dependent on microtubule severing by p60-katanin, dominates after nuclear envelope breakdown, restoring bipolarity.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain

Centrosomes orchestrate microtubule nucleation and spindle assembly during cell division [1,2] and have long been recognized as major anchoring sites for cAMP-dependent protein kinase (PKA) [3,4]. Subcellular compartmentalization of PKA is achieved through the association of the PKA holoenzyme with A-kinase anchoring proteins (AKAPs) [5,6]. AKAPs have been shown to contain a conserved helical motif, responsible for binding to the type II regulatory subunit (RII) of PKA, and a specific targeting motif unique to each anchoring protein that directs the kinase to specific intracellular locations. Here, we show that pericentrin, an integral component of the pericentriolar matrix of the centrosome that has been shown to regulate centrosome assembly and organization, directly interacts with PKA through a newly identified binding domain. We demonstrate that both RII and the catalytic subunit of PKA coimmunoprecipitate with pericentrin isolated from HEK-293 cell extracts and that PKA catalytic activity is enriched in pericentrin immunoprecipitates. The interaction of pericentrin with RII is mediated through a binding domain of 100 amino acids which does not exhibit the structural characteristics of similar regions on conventional AKAPs. Collectively, these results provide strong evidence that pericentrin is an AKAP in vivo.     

Electron Tomography Reveals Novel Microtubule Lattice and Microtubule Organizing Centre Defects in +TIP Mutants

Mal3p and Tip1p are the fission yeast (Schizosaccharomyces pombe) homologues of EB1 and CLIP-170, two conserved microtubule plus end tracking proteins (+TIPs). These proteins are crucial regulators of microtubule dynamics. Using electron tomography, we carried out a high-resolution analysis of the phenotypes caused by mal3 and tip1 deletions. We describe the 3-dimensional microtubule organization, quantify microtubule end structures and uncover novel defects of the microtubule lattices. We also reveal unexpected structural modifications of the spindle pole bodies (SPBs), the yeast microtubule organizing centers. In both mutants we observe an increased SPB volume and a reduced number of MT/SPB attachments. The discovered defects alter previous interpretations of the mutant phenotypes and provide new insights into the molecular functions of the two protein families.     

Assembly of centrosomal proteins and microtubule organization depends on PCM-1

The protein PCM-1 localizes to cytoplasmic granules known as "centriolar satellites" that are partly enriched around the centrosome. We inhibited PCM-1 function using a variety of approaches: microinjection of antibodies into cultured cells, overexpression of a PCM-1 deletion mutant, and specific depletion of PCM-1 by siRNA. All approaches led to reduced targeting of centrin, pericentrin, and ninein to the centrosome. Similar effects were seen upon inhibition of dynactin by dynamitin, and after prolonged treatment of cells with the microtubule inhibitor nocodazole. Inhibition or depletion of PCM-1 function further disrupted the radial organization of microtubules without affecting microtubule nucleation. Loss of microtubule organization was also observed after centrin or ninein depletion. Our data suggest that PCM-1-containing centriolar satellites are involved in the microtubule- and dynactin-dependent recruitment of proteins to the centrosome, of which centrin and ninein are required for interphase microtubule organization.     

The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A

Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.     

Bazooka regulates microtubule organization and spatial restriction of germ plasm assembly in the Drosophila oocyte

Localization of the germ plasm to the posterior of the Drosophila oocyte is required for anteroposterior patterning and germ cell development during embryogenesis. While mechanisms governing the localization of individual germ plasm components have been elucidated, the process by which germ plasm assembly is restricted to the posterior pole is poorly understood. In this study, we identify a novel allele of bazooka (baz), the Drosophila homolog of Par-3, which has allowed the analysis of baz function throughout oogenesis. We demonstrate that baz is required for spatial restriction of the germ plasm and axis patterning, and we uncover multiple requirements for baz in regulating the organization of the oocyte microtubule cytoskeleton. Our results suggest that distinct cortical domains established by Par proteins polarize the oocyte through differential effects on microtubule organization. We further show that microtubule plus-end enrichment is sufficient to drive germ plasm assembly even at a distance from the oocyte cortex, suggesting that control of microtubule organization is critical not only for the localization of germ plasm components to the posterior of the oocyte but also for the restriction of germ plasm assembly to the posterior pole.     

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.     

Centrosomal anchoring of protein kinase C betaII by pericentrin controls microtubule organization, spindle function, and cytokinesis

Location is a critical determinant in dictating the cellular function of protein kinase C (PKC). Scaffold proteins contribute to localization by poising PKC at specific intracellular sites. Using a yeast two-hybrid screen, we identified the centrosomal protein pericentrin as a scaffold that tethers PKC betaII to centrosomes. Co-immunoprecipitation studies reveal that the native proteins interact in cells. Co-overexpression studies show that the interaction is mediated by the C1A domain of PKC and a segment of pericentrin within residues 494-593. Immunofluorescence analysis reveals that endogenous PKC betaII colocalizes with pericentrin at centrosomes. Disruption of this interaction by expression of the interacting region of pericentrin results in release of PKC from the centrosome, microtubule disorganization, and cytokinesis failure. Overexpression of this disrupting fragment has no effect in cells lacking PKC betaII, indicating a specific regulatory role of this isozyme in centrosome function. These results reveal a novel role for PKC betaII in cytokinesis and indicate that this function is mediated by an interaction with pericentrin at centrosomes.