Novel clades of the HU/IHF superfamily point to unexpected roles in the eukaryotic centrosome,chromosome partitioning,and biologic conflicts

The HU superfamily of proteins, with a unique DNA-binding mode, has been extensively studied as the primary chromosome-packaging protein of the bacterial superkingdom. Representatives also play a role in DNA-structuring during recombination events and in eukaryotic organellar genome maintenance. However, beyond these well-studied roles, little is understood of the functional diversification of this large superfamily. Using sensitive sequence and structure analysis methods we identify multiple novel clades of the HU superfamily. We present evidence that a novel eukaryotic clade prototyped by the human CCDC81 protein acquired roles beyond DNA-binding, likely in protein-protein interaction in centrosome organization and as a potential cargo-binding protein in conjunction with Dynein-VII. We also show that these eukaryotic versions were acquired via an early lateral transfer from bacteroidetes, where we predict a role in chromosome partition. This likely happened before the last eukaryotic common ancestor, pointing to potential endosymbiont contributions beyond that of the mitochondrial progenitor. Further, we show that the dramatic lineage-specific expansion of this domain in the bacteroidetes lineage primarily is linked to a functional shift related to potential recognition and preemption of genome invasive entities such as mobile elements. Remarkably, the CCDC81 clade has undergone a similar massive lineage-specific expansion within the archosaurian lineage in birds, suggesting a possible use of the HU superfamily in a similar capacity in recognition of non-self molecules even in this case.     

Centrosome to autophagosome signaling: Specific GABARAP regulation by centriolar satellites

Yeast have one Atg8 protein; however, multiple Atg8 orthologs (LC3s and GABARAPs) are found in humans. We discovered that a population of the Atg8 ortholog GABARAP resides on the centrosome and the peri-centrosomal region. This centrosomal pool of GABARAP translocates to forming autophagosomes upon starvation to activate autophagosome formation in a non-hierarchical pathway. How this centrosome-to-phagophore delivery of GABARAP occurs was not understood. To address this, we have shown that the archetypal centriolar satellite protein PCM1 regulates recruitment of GABARAP to the centrosome. PCM1 recruits GABARAP, but not MAP1LC3B, directly to centriolar satellites through a LC3-interacting region (LIR) motif. Furthermore, PCM1, in concert with its interacting centriolar satellite E3 ligase MIB1, controls GABARAP stability, K48-linked ubiquitination and GABARAP-mediated autophagic flux.     

The carboxy-terminus of protein 4.1r resembles Beta-tubulin

The protein 4.1R is an isoform of a larger family of 4.1 proteins. It is known as a component of the plasma membrane skeleton, but it is also found at the centrosomes in interphase and mitosis. To investigate the properties of the carboxy terminal region of protein 4.1R, we raised antibodies against a peptide representing the last 14 amino acids of 4.1R. These antibodies crossreact with an epitope in beta-tubulin and stain the microtubule network by immunofluorescence. Furthermore, sequence comparison of the carboxy terminal 4.1R peptide sequence with tubulin reveals homology with a region at the end of helix 5 in beta-tubulin, but not alpha-tubulin. A potential function of the 4.1R carboxy terminus in regulating the formation of microtubule networks is discussed.     

In vitro genotoxic effects of ZnO nanomaterials in human peripheral lymphocytes

In this study, possible genotoxic effects of zinc oxide (ZnO) nanoparticles were investigated in cultured human peripheral lymphocytes by using chromosome aberrations and micronucleus assays (MN). For this purpose, the cells were treated with ZnO (1, 2, 5, 10, 15 and 20 μg/mL) for 24 and 48 h. In this research, four types of chromosome aberrations were observed as chromatid and chromosome breaks, fragment and dicentric chromosomes. ZnO induced significant increase of the ratio of chromosomal aberrations as well as percentage of abnormal cells at concentrations of 1, 5, 10 and 20 μg/mL in 24 h treatments. In 48 h treatments, while ZnO nanomaterials induced significant increase of the percentage of abnormal cells only at a concentration of 10 μg/mL, and of chromosome aberration per cell in comparison to the control at concentrations of 5 and 10 μg/mL. On the other hand, this material significantly increased the micronuclei frequency (MN) at concentrations of 10 and 15 μg/mL in comparison to the control. Cytokinesis-block proliferation index was not affected by ZnO treatments. It also decreased the mitotic index in all concentrations at 24 h but not at 48 h. The present results indicate that ZnO nanoparticles are clastogenic, mutagenic and cytotoxic to human lymphocytes in vitro at specific concentrations and time periods.     

Separate to operate: control of centrosome positioning and separation

The centrosome is the main microtubule (MT)-organizing centre of animal cells. It consists of two centrioles and a multi-layered proteinaceous structure that surrounds the centrioles, the so-called pericentriolar material. Centrosomes promote de novo assembly of MTs and thus play important roles in Golgi organization, cell polarity, cell motility and the organization of the mitotic spindle. To execute these functions, centrosomes have to adopt particular cellular positions. Actin and MT networks and the association of the centrosomes to the nuclear envelope define the correct positioning of the centrosomes. Another important feature of centrosomes is the centrosomal linker that connects the two centrosomes. The centrosome linker assembles in late mitosis/G1 simultaneously with centriole disengagement and is dissolved before or at the beginning of mitosis. Linker dissolution is important for mitotic spindle formation, and its cell cycle timing has profound influences on the execution of mitosis and proficiency of chromosome segregation. In this review, we will focus on the mechanisms of centrosome positioning and separation, and describe their functions and mechanisms in the light of recent findings.     

Mitochondrial genome regulates mitotic fidelity by maintaining centrosomal homeostasis

Centrosomes direct spindle morphogenesis to assemble a bipolar mitotic apparatus to enable error-free chromosome segregation and preclude chromosomal instability (CIN). Amplified centrosomes, a hallmark of cancer cells, set the stage for CIN, which underlies malignant transformation and evolution of aggressive phenotypes. Several studies report CIN and a tumorigenic and/or aggressive transformation in mitochondrial DNA (mtDNA)-depleted cells. Although several nuclear-encoded proteins are implicated in centrosome duplication and spindle organization, the involvement of mtDNA encoded proteins in centrosome amplification (CA) remains elusive. Here we show that disruption of mitochondrial function by depletion of mtDNA induces robust CA and mitotic aberrations in osteosarcoma cells. We found that overexpression of Aurora A, Polo-like kinase 4 (PLK4), and Cyclin E was associated with emergence of amplified centrosomes. Supernumerary centrosomes in rho0 (mtDNA-depleted) cells resulted in multipolar mitoses bearing “real” centrosomes with paired centrioles at the multiple poles. This abnormal phenotype was recapitulated by inhibition of respiratory complex I in parental cells, suggesting a role for electron transport chain (ETC) in maintaining numeral centrosomal homeostasis. Furthermore, rho0 cells displayed a decreased proliferative capacity owing to a G₂/M arrest. Downregulation of nuclear-encoded p53 in rho0 cells underscores the importance of mitochondrial and nuclear genome crosstalk and may perhaps underlie the observed mitotic aberrations. By contrast, repletion of wild-type mtDNA in rho0 cells (cybrid) demonstrated a much lesser extent of CA and spindle multipolarity, suggesting partial restoration of centrosomal homeostasis. Our study provides compelling evidence to implicate the role of mitochondria in regulation of centrosome duplication, spindle architecture, and spindle pole integrity.     

Disruption of Clathrin‐Mediated Trafficking Causes Centrosome Overduplication and Senescence

The Hsc70 cochaperone, G cyclin‐associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin‐dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin‐mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin‐dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.     

Msd1/SSX2IP‐dependent microtubule anchorage ensures spindle orientation and primary cilia formation

Anchoring microtubules to the centrosome is critical for cell geometry and polarity, yet the molecular mechanism remains unknown. Here we show that the conserved human Msd1/SSX2IP is required for microtubule anchoring. hMsd1/SSX2IP is delivered to the centrosome in a centriolar satellite‐dependent manner and binds the microtubule‐nucleator γ‐tubulin complex. hMsd1/SSX2IP depletion leads to disorganised interphase microtubules and misoriented mitotic spindles with reduced length and intensity. Furthermore, hMsd1/SSX2IP is essential for ciliogenesis, and during zebrafish embryogenesis, knockdown of its orthologue results in ciliary defects and disturbs left‐right asymmetry. We propose that the Msd1 family comprises conserved microtubule‐anchoring proteins.     

氯苯胁迫对蚕豆幼苗生长和细胞分裂的影响

研究了1,2,4-三氯苯(TCB)对蚕豆幼苗生长、根尖细胞分裂及染色体畸变的影响．结果表明,随TCB浓度增加和处理时间延长,蚕豆幼苗根长的生长及根尖细胞有丝分裂指数降低甚至停止．TCB诱发蚕豆根尖细胞有丝分裂过程中染色体数目畸变和结构畸变．50-100μg.g^-1TCB胁迫12-24h,蚕豆根尖染色体的主要损伤形式为c-有丝分裂、染色体桥和不均匀排列,其出现百分率达1．0％--10.3％．300μg.g^-1TCB胁迫12-96h,蚕豆根尖细胞中染色体粘连(S)、S+染色体断裂(S+B)、S+染色体环(S+R)、S+染色体不均匀排列(S+A)及S+染色体桥(S+Be)出现的百分率达47．9％--88.9％,各种类型染色体断裂出现的百分率仅为18．1％--29．6％,说明蚕豆根尖细胞染色体畸变分析可作为TCB土壤污染监测的敏感生物监测指标．     

无精和严重少精症患者的遗传缺陷分析——-附2例世界首报染色体异常核型

对217例无精和严重少精症患者外周血淋巴细胞染色体核型进行分析,并采用聚合酶链反应对7例Y染色体结构异常患者的AZFc区进行检测。发现187例无精症患者中检出异常核型77例（41.18%）（其中46,XY,t(6;14)(p21;p13),46,XY,t(8;12)(p21;q24)为世界首报核型）,主要涉及染色体异常（数目异常和结构异常）;染色体异态（Y染色体异态和9号染色体臂间倒位）及46,XX性反转;30例严重少精症患者中检出异常核型4例（13.33%）（结构异常和46,XX性反转）。由此可见,性染色体数目和结构异常是精子发生障碍的主要原因,其次常染色体的某些断裂点也可能影响精子发生。AZFc区的缺失与否与精子发生也有直接关系。