Ccdc103 promotes myeloid cell proliferation and migration independent of motile cilia

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show Coiled-coil domain containing protein 103 (CCDC103), one of the genes that when mutated is known to cause PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 is expressed in human myeloid cells, where it co-localizes with cytoplasmic microtubules. Zebrafish ccdc103/schmalhans (smh) mutants have macrophages and neutrophils with reduced proliferation, abnormally-rounded cell morphology, and an inability to migrate efficiently to the site of sterile wounds, all of which are consistent with a loss of cytoplasmic microtubule stability. Furthermore, we demonstrate that direct interactions between CCDC103 and Sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects observed in smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients.

Analysis of Water-Soluble Proteins by Two-Dimensional Electrophoresis in the Encystment Process of Colpoda cucullus Nag-1 and Cytoskeletal Dynamics

Assays of protein contained in water-soluble fraction of encysting cells Colpoda cucullus Nag-1 by two-dimensional electrophoresis (2-D PAGE) and mass spectrometry (MS) revealed that the amount of β-tubulin abruptly increased in 2.5–10 h after encystment induction. Judging from the results that total α-tubulin content did not decrease much until 12 h after encystment induction, the result indicates that disassembly of microtubules may occur soon after encystment is induced. Therefore, we tried to visualize dynamics of microtubules. Immunofluorescence microscopy using anti-α-tubulin antibody indicated that disassembly of axonemal microtubules of cilia became within 1.5 h after encystment induction, and resorbed in 3 days. Although the cytoplasmic microtubules failed to be visualized clearly, encystmentdependent globulation of cells was promoted by taxol, an inhibitor of disassembly of microtubules. It is possible that a temporary formation of cytoplasmic microtubules may be involved in cell globulation. The phosphorylation level of actin (43 kDa) became slightly elevated just after encystment induction. Lepidosomes, the sticky small globes surrounding encysting cells, were vividly stained with Acti-stain 555 phalloidin, suggesting that 43-kDa actin or its homologues may be contained in lepidosomes.

Cilia-independent requirements for Ccdc103 promoting proliferation and migration of myeloid cells

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show Coiled-coiled domain containing protein 103 (CCDC103), mutations in which underlie PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 co-localizes with cytoplasmic microtubules in human myeloid cells. Zebrafish ccdc103/schmalhans (smh) mutants have reduced macrophage and neutrophil proliferation, rounded cell morphology, and an inability to migrate efficiently to the site of sterile wounds. Furthermore, we demonstrate that direct interactions between CCDC103 and Sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects of smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients.Summary StatementWe show Ccdc103 regulates myeloid migration and proliferation independent of cilia in zebrafish and that mutations in CCDC103 that cause primary ciliary dyskinesia abrogate interactions with the microtubule-stabilizing protein SPAG6.

Arp2/3 complex-mediated actin assembly drives microtubule-independent motility and phagocytosis in the evolutionarily divergent amoeba Naegleria

ABSTRACTMuch of our current understanding of actin-driven phenotypes in eukaryotes has come from the “yeast to human” opisthokont lineage, as well as the related amoebozoa. Outside of these groups lies the genus Naegleria, which shared a common ancestor with humans over a billion years ago, and includes the deadly “brain-eating amoeba.” Unlike nearly every other known eukaryotic cell type, Naegleria amoebae are thought to lack cytoplasmic microtubules. The absence of microtubules suggests that these amoebae rapidly crawl and phagocytose bacteria using actin alone. Although this makes Naegleria a powerful system to probe actin-driven functions in the absence of microtubules, surprisingly little is known about Naegleria’s actin cytoskeleton. Here, we use microscopy and genomic analysis to show that Naegleria amoebae have an extensive actin cytoskeletal repertoire, complete with nucleators and nucleation promoting factors. Naegleria use this cytoskeletal machinery to generate Arp2/3-dependent lamellar protrusions, which correlate with the capacity to migrate and phagocytose bacteria. Because human cells also use Arp2/3-dependent lamellar protrusions for motility and phagocytosis, this work supports an evolutionarily ancient origin for these actin-driven processes and establishes Naegleria as a natural model system for studying microtubule-independent cytoskeletal phenotypes.

Centrioles and Ciliary Structures during Male Gametogenesis in Hexapoda: Discovery of New Models

Centrioles are-widely conserved barrel-shaped organelles present in most organisms. They are indirectly involved in the organization of the cytoplasmic microtubules both in interphase and during the cell division by recruiting the molecules needed for microtubule nucleation. Moreover, the centrioles are required to assemble cilia and flagella by the direct elongation of their microtubule wall. Due to the importance of the cytoplasmic microtubules in several aspects of the cell life, any defect in centriole structure can lead to cell abnormalities that in humans may result in significant diseases. Many aspects of the centriole dynamics and function have been clarified in the last years, but little attention has been paid to the exceptions in centriole structure that occasionally appeared within the animal kingdom. Here, we focused our attention on non-canonical aspects of centriole architecture within the Hexapoda. The Hexapoda is one of the major animal groups and represents a good laboratory in which to examine the evolution and the organization of the centrioles. Although these findings represent obvious exceptions to the established rules of centriole organization, they may contribute to advance our understanding of the formation and the function of these organelles.

Nuclear Mechanics in the Fission Yeast

In eukaryotic cells, the organization of the genome within the nucleus requires the nuclear envelope (NE) and its associated proteins. The nucleus is subjected to mechanical forces produced by the cytoskeleton. The physical properties of the NE and the linkage of chromatin in compacted conformation at sites of cytoskeleton contacts seem to be key for withstanding nuclear mechanical stress. Mechanical perturbations of the nucleus normally occur during nuclear positioning and migration. In addition, cell contraction or expansion occurring for instance during cell migration or upon changes in osmotic conditions also result innuclear mechanical stress. Recent studies in Schizosaccharomyces pombe (fission yeast) have revealed unexpected functions of cytoplasmic microtubules in nuclear architecture and chromosome behavior, and have pointed to NE-chromatin tethers as protective elements during nuclear mechanics. Here, we review and discuss how fission yeast cells can be used to understand principles underlying the dynamic interplay between genome organization and function and the effect of forces applied to the nucleus by the microtubule cytoskeleton.

The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72–γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

Actin-dependent regulation of cilia length by the inverted formin FHDC1

A primary cilium is found on most mammalian cells, where it acts as a cellular antenna for the reception of both mechanical and chemical signals. A variety of diseases are associated with defective ciliogenesis, reflecting the ubiquity of the function of cilia and the number of proteins required for their assembly. Proper cilia length is necessary for cilia signaling and is regulated through a poorly understood balance of assembly and disassembly rates. FHDC1 is a unique member of the formin family of cytoskeletal regulatory proteins. Overexpression of FHDC1 induces F-actin accumulation and microtubule stabilization and acetylation. We find that overexpression of FHDC1 also has profound effects on ciliogenesis; in most cells FHDC1 overexpression blocks cilia assembly, but the cilia that are present are immensely elongated. FHDC1-induced cilia growth requires the FHDC1 FH2 and microtubule-binding domain and results from F-actin–dependent inhibition of cilia disassembly. FHDC1 depletion, or treatment with a pan-formin inhibitor, inhibits cilia assembly and induces cilia resorption. Endogenous FHDC1 protein localizes to cytoplasmic microtubules converging on the base of the cilia, and we identify the subdistal appendage protein Cep170 as an FHDC1 interacting protein. Our results suggest that FHDC1 plays a role in coordinating cytoskeletal dynamics during normal cilia assembly.

Polo-like kinase Cdc5 regulates Spc72 recruitment to spindle pole body in the methylotrophic yeast Ogataea polymorpha

Cytoplasmic microtubules (cMT) control mitotic spindle positioning in many organisms, and are therefore pivotal for successful cell division. Despite its importance, the temporal control of cMT formation remains poorly understood. Here we show that unlike the best-studied yeast Saccharomyces cerevisiae, position of pre-anaphase nucleus is not strongly biased toward bud neck in Ogataea polymorpha and the regulation of spindle positioning becomes active only shortly before anaphase. This is likely due to the unstable property of cMTs compared to those in S. cerevisiae. Furthermore, we show that cMT nucleation/anchoring is restricted at the level of recruitment of the γ-tubulin complex receptor, Spc72, to spindle pole body (SPB), which is regulated by the polo-like kinase Cdc5. Additionally, electron microscopy revealed that the cytoplasmic side of SPB is structurally different between G1 and anaphase. Thus, polo-like kinase dependent recruitment of γ-tubulin receptor to SPBs determines the timing of spindle orientation in O. polymorpha.

Effect of Sublethal Nickel Chloride Exposure on Crayfish, Astacus leptodactylus Ovary: An Ultrastructural, Autometallographic, and Electrophoretic Analyses

Cytological responses in different organs of sentinel organisms have proven to be useful tools for characterizing the health status of those organisms and assessing the impact of environmental contaminants. Our study shows that nickel (II) accumulated in both germ cells (oogonia and developing oocytes) and somatic cells (muscle cells, follicle cells) in the Astacus leptodactylus ovary. Muscle cells from ovarian wall show disorganization and the disruption of cytoplasmic microtubules and pyknosis of the cell nucleus. Follicle cells, both those that surround the developing oocytes and also those that are not associated with the oocytes contained within the cytoplasm vacuoles of different sizes, degenerated mitochondria, myelin bodies, disorganized microtubules, and pyknotic nuclei. The most evident pathological phenomenon was the alteration and disorganization of the basal matrix, which separates the ovarian interstitium from ovarian follicles compartment. Exposure to nickel induces cytoplasmic vacuolation in oogonia and developing oocytes, structural alteration of the developing yolk granules and condensation of the nucleoli. Ultrastructural autometallography has shown grains of silver-enhanced nickel inside the cytoplasm of the muscle cells with altered morphology, including the cytoplasm, nucleus, and basal matrix of the follicle cells, and in intracisternal granules and developing yolk granules of the oocytes.