Geminin Coordinates Cell Cycle and Developmental Control

Growth and differentiation are two major themes in embryonic development. Numerous cell divisions have to be regulated on the path from a unicellular embryo, the zygote, to the multicellular structures of a mature being. Numerous functions, specializations and cellular identities have to be generated, in order to form a complex and mature animal. Numerous mechanisms have to control the correct assignment and acquisition of cellular fates, as well as the right timing and allocation of cells. Therefore, a strict coordination has to occur between embryonic patterning and the cell cycle. From this point of view, dual roles or mutual interactions of typical proliferation and developmental control genes are likely. Recently, new light was shed on these issues by identifying the nuclear protein Geminin as a molecular coordinator between the cell cycle and axial patterning. We summarize the role of Geminin in cell cycle, in the embryonic patterning controlled by Hox genes, providing insights into cell cycle regulators in embryonic development, and, conversely, typical developmental control genes in cell cycle regulation.     

The Centrosome and Its Duplication Cycle

The centrosome was discovered in the late 19th century when mitosis was first described. Long recognized as a key organelle of the spindle pole, its core component, the centriole, was realized more than 50 or so years later also to comprise the basal body of the cilium. Here, we chart the more recent acquisition of a molecular understanding of centrosome structure and function. The strategies for gaining such knowledge were quickly developed in the yeasts to decipher the structure and function of their distinctive spindle pole bodies. Only within the past decade have studies with model eukaryotes and cultured cells brought a similar degree of sophistication to our understanding of the centrosome duplication cycle and the multiple roles of this organelle and its component parts in cell division and signaling. Now as we begin to understand these functions in the context of development, the way is being opened up for studies of the roles of centrosomes in human disease.     

The Coordination of Centrosome Reproduction with Nuclear Events of the Cell Cycle in the Sea Urchin Zygote

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1–cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Δ-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase–anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary “licensing” event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.     

ZNF403，一个新的细胞周期调节因子的功能研究

ZNF403和LCRG1是人类基因ZNF403的2个不同转录剪切本.以往的研究表明LCRG1在喉癌细胞株Hep-2中具有抑瘤特性.本研究旨在探明ZNF403和LCRG1不同剪切本之间的关系以及在肿瘤细胞中对ZNF403的功能进行研究.首先,采用实时荧光定量PCR对这2个转录本的相对表达水平进行分析,结果表明,ZNF403表达水平在不同细胞株中明显高于LCRG1(>10倍),为该基因的主要转录表达产物.随后分别采用MTT细胞生长分析法和裸鼠体内成瘤实验在体外和体内对ZNF403的功能进行分析,结果显示ZNF403的基因沉默可以同时在体内和体外抑制喉癌细胞Hep-2细胞的生长.为了探明其作用机制,本研究还采用细胞信息学、流式细胞周期分析术和高通量PCR点阵分析方法进一步分析,结果表明,ZNF403的基因沉默可显著抑制细胞DNA的复制并延缓细胞周期进入到有丝分裂期.同时发现ZNF403可调节一系列的细胞周期调节蛋白如MCM2、p21、ATM、MRE11A等.综上研究提示ZNF403为一新的细胞周期调节因子,其功能的缺失与肿瘤发生发展密切相关.     

Macrophage Migration Inhibitory Factor Coordinates DNA Damage Response with the Proteasomal Control of the Cell Cycle

Proper repair of DNA damage is critical for protecting genomic stability, cellular viability and suppression of tumorigenesis. Both p53-dependent and p53-independent pathways have evolved to coordinate the cellular response following DNA damage. In this review, we highlight the importance of the ubiquitously expressed protein macrophage migration inhibitory factor (MIF) for an appropriate response to DNA damage. We discuss the mechanisms by which MIF affects the activity of the ubiquitin-proteasome system, and how this impacts on the integrity of the genome and on cancer.     

A Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role

The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.Apicomplexan parasites cause a multitude of illnesses through infection of both human and livestock hosts. Members of this phylum include the opportunistic human parasites Toxoplasma gondii and Cryptosporidium parvum, pathogens of livestock, including Theileria annulata and Eimeria tenalla, and most notably the Plasmodium species, the causative agents of malaria in humans. Infection with P. falciparum results in ∼1–3 million deaths and a further 500 million infections annually (1).During various stages of the Apicomplexan lifecycle the parasites require motility to migrate through their insect and vertebrate hosts and to invade and internalize themselves within targeted host cells (2–4). The parasite''s unique mechanism of gliding motility is powered by an Apicomplexan-specific motor complex termed the actin-myosin motor (5), which resides between the outer plasma membrane and inner membrane complex (IMC)⁴ (6). The IMC is a continuous patchwork of flattened vesicular cisternae located directly beneath the plasma membrane and overlying the cytoskeletal network (7, 8). The IMC appears to arise from Golgi-associated vesicles flattened during parasite maturation to form large membranous sheets, which envelope the parasite and leave only a small gap at the extreme parasite apex (9).The myosin component of the actin-myosin motor has previously been defined as a tetrameric complex consisting of a class XIV myosin termed Myo-A (10), a myosin tail interacting protein (also called myosin light chain) (7) and the two glideosome-associated proteins GAP45 and GAP50 (11). These motor components are linked to the outer IMC membrane via the membrane proteins GAP45/50 (11). Between the plasma membrane and the IMC are actin filaments held in place through aldolase-mediated contact with the C-terminal tails of plasma membrane-spanning adhesive proteins whose ectodomains bind substrate and host cells (2). To power the forward movement of apicomplexan zoite stages, myosin pulls the actin filaments and their attached adhesins rearward. For this to succeed the GAP-myosin complex must presumably be fixed to the IMC, possibly via interactions with unidentified proteins linking the motor to the underlying cytoskeleton. Studies of fluorescently tagged GAP50 confirm it is relatively immobile within the IMC, however attempts to identify potential anchoring proteins have not been successful and have instead indicated that GAP50 may be immobilized by the lipid-raft like properties of the IMC membranes (12).The actin-myosin complex is confined to the outer IMC membrane while the opposing innermost IMC membrane is studded with 9 nm intramembranous particles, revealed by electron microscopy of freeze fractured Toxoplasma tachyzoites and Plasmodium ookinetes (13, 14). The size of these particles suggests that the proteins involved are likely to form high molecular weight complexes that overlay the parasite''s cytoskeletal network and possibly anchor the IMC to the cytoskeleton (12–15). Due to the close apposition of the inner and outer IMC membranes (14, 16), it is possible that the intramembranous particles could bridge the IMC lumen and interact with the GAP-myosin complex contributing to its stabilization within the IMC.To identify putative proteins that might be components of the intramembranous particles, we examined data from the detergent-resistant membrane (DRM) proteome of schizont-stage P. falciparum parasites containing developing merozoites (17, 18). DRMs, or lipid-rafts, were of considerable interest, because they appeared to harbor proteins involved in host cell invasion such as glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins. Our data also indicated that P. falciparum schizont-stage DRMs contained the IMC proteins PfGAP45/50 (17), and recent studies in T. gondii have also suggested that the IMC is enriched in DRMs (12). Another study indicated that when P. falciparum DRM protein complexes were separated by blue native gel electrophoresis, a band was produced containing PfGAP45/50 and PfMyo-A as well as a novel six-pass transmembrane protein (PlasmoDB: PFD1110w, GenBank^TM: {"type":"entrez-protein","attrs":{"text":"CAD49269","term_id":"23498297","term_text":"CAD49269"}}CAD49269) (18). This protein was related to another six-pass transmembrane DRM protein (PlasmoDB: MAL13P1.130, GenBank^TM: {"type":"entrez-protein","attrs":{"text":"CAD52385","term_id":"23615394","term_text":"CAD52385"}}CAD52385) we had previously identified in P. falciparum schizont-stage DRMs (17).We show here that MAL13P1.130 and PFD1110w, termed PfGAPM1 and PfGAPM2 (glideosome-associated protein with multiple-membrane spans), respectively, belong to a family of proteins specific to the Apicomplexa and demonstrate that P. falciparum GAPM proteins, and their orthologues in T. gondii, localize to the parasite IMC. The GAPMs form high molecular weight complexes that are resistant to dissociation and solubilization by a variety of common detergents and could therefore be components of the intramembranous particles seen in electron microscopy. When isolated by immunoprecipitation, the GAPM complexes co-purify with components of the actin-myosin motor and particularly the parasite cytoskeletal network suggesting GAPMs could anchor the IMC to the cytoskeleton and perhaps even play a role in tethering the motor to cytoskeleton.     

A Novel Cell Cycle Inhibitor Stalls Replication Forks and Activates S Phase Checkpoint

DNA replication checkpoint is activated in response to replication stresses. It maintains the integrity of stalled replication forks and prevents premature segregation of largely unreplicated chromosomes. In budding yeast, Mec1 and Rad53 kinases (homologous to mammalian ATM/ATR and Chk2 kinases, respectively) are the main effectors of this checkpoint control. Using a yeast based screen, we have identified acompound (named here ENA) which inhibits DNA replication and activatesMec1/Rad53 checkpoint. A brief exposure to this compound stops fork progression at or near replication origin and renders the forks incompetent to resume replication despite the presence of a functional checkpoint. ENA also inhibits DNA synthesis in mammalian cells leading to the activation of ATM/ATR pathway and the induction of apoptosis in a p53 independent manner. Interestingly, ENA acts as an effective antiproliferative agent against a subset of cancer cell lines and as an anti-tumor agent against human xenografts in mice. Thus, ENA is a potent cell cycle inhibitor with conceivable therapeutic potential.     

Novel Functions for Cell Cycle Genes in Nervous System Development

Many cell cycle genes are known to play important roles in regulating proliferation in the nervous system, however, a growing body of research has proposed that these genes have diverse functions beyond cell cycle regulation. Through the study of new genetic models, cell cycle regulatory genes have been shown to impact on a number of processes during nervous system development including apoptosis, differentiation, and, most recently, neuronal migration. Here we emphasize that the proposed roles for cell cycle genes in neuronal differentiation and migration are not the consequence of deregulated cell cycle, but represent truly novel functions for cell cycle genes.     

Ska2/FAM33A:一个参与细胞周期调控与肿瘤发生的新基因

Ska2(spindle and KT associated 2),也称FAM33A(family with sequence similarity 33,member A),是一个最近鉴定的参与细胞周期调控与肿瘤发生发展的新基因。现有研究初步证实,Ska2参与组成Ska复合体,在有丝分裂中期纺锤体检验点关闭中起重要作用;Ska2在小细胞肺癌和乳腺癌中呈现表达上调,可通过糖皮质激素受体等途径参与细胞增殖调节和肿瘤发生发展;NF-κΒ和CREB等转录因子可能参与Ska2的表达调控。Ska2有望成为一个恶性肿瘤诊断和靶向治疗的新靶点。     

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements

Apicomplexan parasites generally invade their host cells by anchoring the parasite to the host membrane through a structure called the moving junction (MJ). This MJ is also believed to sieve host proteins from the nascent parasitophorous vacuole membrane, which likely protects the pathogen from lysosomal destruction. Previously identified constituents of the Toxoplasma MJ have orthologues in Plasmodium , indicating a conserved structure throughout the Apicomplexa. We report here two novel MJ proteins, RON5 and RON8. While RON5 is conserved in Plasmodium , RON8 appears restricted to the coccidia. RON8, which is likely essential, co-immunoprecipitates RON5 and known MJ proteins from extracellular parasites, indicating that a preformed complex exists within the parasites. Upon secretion, we show that RON8 within the MJ localizes to the cytoplasmic face of the host plasma membrane. To examine interactions between RON8 and the host cell, we expressed RON8 in mammalian cells and show that it targets to its site of action at the periphery in a manner dependent on the C-terminal portion of the protein. The discovery of RON5 and RON8 provides new insight into conserved and unique elements of the MJ, furthering our understanding of how the MJ contributes to the intricate mechanism of Apicomplexan invasion.