Christophe Lefebvre

Recruitment of Mad2 to the Kinetochore Requires the Rod/Zw10 Complex

Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.

Meiotic spindle stability depends on MAPK-interacting and spindle-stabilizing protein (MISS), a new MAPK substrate

Vertebrate oocytes arrest in the second metaphase of meiosis (metaphase II [MII]) by an activity called cytostatic factor (CSF), with aligned chromosomes and stable spindles. Segregation of chromosomes occurs after fertilization. The Mos/…/MAPK (mitogen-activated protein kinases) pathway mediates this MII arrest. Using a two-hybrid screen, we identified a new MAPK partner from a mouse oocyte cDNA library. This protein is unstable during the first meiotic division and accumulates only in MII, where it localizes to the spindle. It is a substrate of the Mos/…/MAPK pathway. The depletion of endogenous RNA coding for this protein by three different means (antisense RNA, double-stranded [ds] RNA, or morpholino oligonucleotides) induces severe spindle defects specific to MII oocytes. Overexpressing the protein from an RNA not targeted by the morpholino rescues spindle destabilization. However, dsRNA has no effect on the first two mitotic divisions. We therefore have discovered a new MAPK substrate involved in maintaining spindle integrity during the CSF arrest of mouse oocytes, called MISS (for MAP kinase–interacting and spindle-stabilizing protein).

In Vivo Dynamics of the Rough Deal Checkpoint Protein during Drosophila Mitosis

Rough Deal (Rod) and Zw10 are components of a complex required for the metazoan metaphase checkpoint and for recruitment of dynein/dynactin to the kinetochore. The Rod complex, like most classical metaphase checkpoint components, forms part of the outer domain of unattached kinetochores. Here we analyze the dynamics of a GFP-Rod chimera in living syncytial Drosophila embryos. Uniquely among checkpoint proteins, GFP-Rod robustly streams from kinetochores along microtubules, from the time of chromosome attachment until anaphase onset. Prometaphase and metaphase kinetochores continuously recruit new Rod, thus feeding the current. Rod flux from kinetochores appears to require biorientation but not tension because it continues in the presence of taxol. As with Mad2, kinetochore- and spindle-associated Rod rapidly turns over with free cytosolic Rod, both during normal mitosis and after colchicine treatment, with a t1/2 of 25-45 s. GFP-Rod coimmunoprecipitates with dynein/dynactin, and in the absence of microtubules both Rod and dynactin accumulate on kinetochores. Nevertheless, Rod and dynein/dynactin behavior are distinguishable. We propose that the Rod complex is a major component of the fibrous corona and that the recruitment of Rod during metaphase is required to replenish kinetochore dynein after checkpoint conditions have been satisfied but before anaphase onset.

DOC1R: a MAP kinase substrate that control microtubule organization of metaphase II mouse oocytes

For the success of fertilization, spindles of vertebrate oocytes must remain stable and correctly organized during the arrest in metaphase II of meiosis. Using a two-hybrid screen with MAPK as a bait, we have recently identified MISS (MAPK interacting and spindle stabilizing) which controls mouse oocyte metaphase II spindle stability. Using the same screen, we identify another MAPK partner, DOC1R (Deleted in oral cancer one related), a murine homologue of a potential human tumor suppressor gene. We characterize DOC1R during mouse oocyte meiosis resumption. DOC1R is regulated by phosphorylation during meiotic maturation by MPF (M-phase promoting factor) and by the MOS/.../MAPK pathway. DOC1R and a DOC1R-GFP fusion localize to microtubules during meiotic maturation. Consistent with this microtubular localization, we show, by antisense and double-stranded RNA injection, that depletion of DOC1R induces microtubule defects in metaphase II oocytes. These defects are rescued by overexpressing a Xenopus DOC1R, showing that they are specific to DOC1R. Thus, the discovery of DOC1R, a substrate of MAPK that regulates microtubule organization of metaphase II mouse oocytes, reinforces the importance of this pathway in the control of spindle stability during the metaphase II arrest.

CeVPS‐27 is an Endosomal Protein Required for the Molting and the Endocytic Trafficking of the Low‐Density Lipoprotein Receptor‐Related Protein 1 in Caenorhabditis elegans

Class E vacuolar protein‐sorting (Vps) proteins were first described in yeast as being involved in receptor‐mediated endocytosis and multivesicular body formation. Inactivation by RNA interference of the class E VPS genes of the nematode Caenorhabditis elegans revealed heterogeneous phenotypes. We have further characterized the role of the essential gene Cevps‐27, ortholog of human hepatocyte growth factor‐regulated tyrosine kinase substrate, during the development of C. elegans. Use of green fluorescent protein fusion constructs and antibody staining revealed that Cevps‐27 localizes to endosomal membranes. It is widely expressed but enriched in epithelial cells. Cevps‐27 mutants presented enlarged endosomal structures and an accumulation of autophagic vesicles as revealed by electron microscopy and the analysis of the autophagic marker LGG‐1. Cevps‐27 animals arrested at L2‐L3 molt with an inability to degrade their old cuticle. This molting phenotype was more severe when Cevps‐27 worms were grown on suboptimal concentrations of cholesterol. Furthermore, defective endocytic trafficking of the low‐density lipoprotein receptor‐related protein 1 (LRP‐1) was also observed in Cevps‐27 mutants. These results indicate that CeVPS‐27 is required for endosomal and autophagic pathways in C. elegans and plays a crucial role in the control of molting through LRP‐1 internalization and cholesterol traffic.

Separating the spindle, checkpoint, and timer functions of BubR1.

The BubR1 kinase domain controls spindle attachment to the kinetochores, whereas the KEN domain regulates activation of the spindle assembly checkpoint.

Evidence for a novel chemotactic C1q domain-containing factor in the leech nerve cord.

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells including microglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by interacting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fully repair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglial cells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologous to vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neurons and glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q. The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors (Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR in C1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies, HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglial cells as human C1q. In summary, we have identified, for the first time, a molecule homologous to mammalian C1q in leech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better understand leech CNS repair mechanisms.

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

A homologous form of human interleukin 16 is implicated in microglia recruitment following nervous system injury in leech Hirudo medicinalis

In contrast to mammals, the medicinal leech Hirudo medicinalis can completely repair its central nervous system (CNS) after injury. This invertebrate model offers unique opportunities to study the molecular and cellular basis of the CNS repair processes. When the leech CNS is injured, microglial cells migrate and accumulate at the site of lesion, a phenomenon known to be essential for the usual sprouting of injured axons. In the present study, we demonstrate that a new molecule, designated HmIL‐16, having functional homologies with human interleukin‐16 (IL‐16), has chemotactic activity on leech microglial cells as observed using a gradient of human IL‐16. Preincubation of microglial cells either with an anti‐human IL‐16 antibody or with anti‐HmIL‐16 antibody significantly reduced microglia migration induced by leech‐conditioned medium. Functional homology was demonstrated further by the ability of HmIL‐16 to promote human CD4+ T cell migration which was inhibited by antibody against human IL‐16, an IL‐16 antagonist peptide or soluble CD4. Immunohistochemistry of leech CNS indicates that HmIL‐16 protein present in the neurons is rapidly transported and stored along the axonal processes to promote the recruitment of microglial cells to the injured axons. To our knowledge, this is the first identification of a functional interleukin‐16 homologue in invertebrate CNS. The ability of HmIL‐16 to recruit microglial cells to sites of CNS injury suggests a role for HmIL‐16 in the crosstalk between neurons and microglia in the leech CNS repair.

The ESCRT-III protein CeVPS-32 is enriched in domains distinct from CeVPS-27 and CeVPS-23 at the endosomal membrane of epithelial cells

Background information. Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs.