Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 µm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia.

Cilia and flagella are evolutionarily conserved structures that play various physiological roles in diverse cell types. Defects in motile cilia result in primary ciliary dyskinesia (PCD), the most prominent ciliopathy, characterized by the association of respiratory symptoms, male infertility, and, in nearly 50% of cases, situs inversus. So far, most identified disease-causing mutations involve genes encoding various ciliary components, such those belonging to the dynein arms that are essential for ciliary motion. Following a candidate-gene approach based on data from a mutant strain of the biflagellated alga Chlamydomonas reinhardtii carrying an ODA7 defect, we identified four families with a PCD phenotype characterized by the absence of both dynein arms and loss-of-function mutations in the human orthologous gene called LRRC50. Functional analyses performed in Chlamydomonas reinhardtii and in another flagellated protist, Trypanosoma brucei, support a key role for LRRC50, a member of the leucine-rich-repeat superfamily, in cytoplasmic preassembly of dynein arms.

Pericentriolar matrix (PCM) integrity relies on cenexin and polo-like kinase (PLK)1.

Polo-like-kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa), zebrafish embryos, and phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation by maintaining PCM cohesion in a PLK1-dependent manner. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to sequester PLK1 that limits PCM substrate phosphorylation events required for PCM cohesion.

Rab11 endosomes and Pericentrin coordinate centrosome movement during pre-abscission in vivo.

The last stage of cell division involves two daughter cells remaining interconnected by a cytokinetic bridge that is cleaved during abscission. Conserved between the zebrafish embryo and human cells, we found that the oldest centrosome moves in a Rab11-dependent manner towards the cytokinetic bridge sometimes followed by the youngest. Rab11-endosomes are organized in a Rab11-GTP dependent manner at the mother centriole during pre-abscission, with Rab11 endosomes at the oldest centrosome being more mobile compared with the youngest. The GTPase activity of Rab11 is necessary for the centrosome protein, Pericentrin, to be enriched at the centrosome. Reduction in Pericentrin expression or optogenetic disruption of Rab11-endosome function inhibited both centrosome movement towards the cytokinetic bridge and abscission, resulting in daughter cells prone to being binucleated and/or having supernumerary centrosomes. These studies suggest that Rab11-endosomes contribute to centrosome function during pre-abscission by regulating Pericentrin organization resulting in appropriate centrosome movement towards the cytokinetic bridge and subsequent abscission.

Chromosome misalignment is associated with PLK1 activity at cenexin-positive mitotic centrosomes.

The mitotic kinase, polo-like kinase 1 (PLK1), facilitates the assembly of the two mitotic spindle poles, which are required for the formation of the microtubule-based spindle that ensures appropriate chromosome distribution into the two forming daughter cells. Spindle poles are asymmetric in composition. One spindle pole contains the oldest mitotic centriole, the mother centriole, where the majority of cenexin, the mother centriole appendage protein and PLK1 binding partner, resides. We hypothesized that PLK1 activity is greater at the cenexin-positive older spindle pole. Our studies found that PLK1 asymmetrically localizes between spindle poles under conditions of chromosome misalignment, and chromosomes tend to misalign toward the oldest spindle pole in a cenexin- and PLK1-dependent manner. During chromosome misalignment, PLK1 activity is increased specifically at the oldest spindle pole, and this increase in activity is lost in cenexin-depleted cells. We propose a model where PLK1 activity elevates in response to misaligned chromosomes at the oldest spindle pole during metaphase.

Gravin regulates centrosome function through PLK1.

We propose to understand how the mitotic kinase PLK1 drives chromosome segregation errors, with a specific focus on Gravin, a PLK1 scaffold. In both three-dimensional primary prostate cancer cell cultures that are prone to Gravin depletion and Gravin short hairpin RNA (shRNA)-treated cells, an increase in cells containing micronuclei was noted in comparison with controls. To examine whether the loss of Gravin affected PLK1 distribution and activity, we utilized photokinetics and a PLK1 activity biosensor. Gravin depletion resulted in an increased PLK1 mobile fraction, causing the redistribution of active PLK1, which leads to increased defocusing and phosphorylation of the mitotic centrosome protein CEP215 at serine-613. Gravin depletion further led to defects in microtubule renucleation from mitotic centrosomes, decreased kinetochore-fiber integrity, increased incidence of chromosome misalignment, and subsequent formation of micronuclei following mitosis completion. Murine Gravin rescued chromosome misalignment and micronuclei formation, but a mutant Gravin that cannot bind PLK1 did not. These findings suggest that disruption of a Gravin-PLK1 interface leads to inappropriate PLK1 activity contributing to chromosome segregation errors, formation of micronuclei, and subsequent DNA damage.

Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes.

The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly.

Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia.

Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins.

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.

Gender differences in cardiac ACE expression are normalized in androgen-deprived male mice.

Gender differences have been described in the response of the cardiovascular system to a number of stimuli, including ventricular remodeling in response to pressure overload, but the molecular basis for these differences remains unclear. Because gender differences in the cardiac expression of angiotensin-converting enzyme (ACE) could contribute to differences in myocardial remodeling, we examined myocardial ACE expression in age-matched male and female mice. Ventricular ACE was more abundant in male than female mice at both mRNA and protein levels. These differences became apparent once the mice reached sexual maturity and became more pronounced with increasing age. The influence of mouse gonadal status on ventricular ACE expression was also examined. Oophorectomy slightly increased ACE levels in female mice, whereas ventricular ACE levels were substantially decreased in androgen-deprived males. The antithetical changes in ventricular ACE abundance seen in agonadal male and female mice suggest that testosterone as well as estrogen may play a role in regulating ACE expression in the heart.