RZZ-Spindly and CENP-E form an integrated platform to recruit dynein to the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

A mechanism that integrates microtubule motors of opposite polarity at the kinetochore corona.

Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin 7) and Dynein-Dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we report near-complete depletion of RZZS and DD from kinetochores after depletion of CENP-E and the outer kinetochore protein KNL1. With inhibited MPS1, CENP-E, which we show binds directly to RZZS, is required to retain kinetochore RZZS. An RZZS phosphomimetic mutant bypasses this requirement. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.

Regulators of tubulin polyglutamylation control nuclear shape and cilium disassembly by balancing microtubule and actin assembly.

Cytoskeletal networks play an important role in regulating nuclear morphology and ciliogenesis. However, the role of microtubule (MT) post-translational modifications in nuclear shape regulation and cilium disassembly has not been explored. Here we identified a novel regulator of the tubulin polyglutamylase complex (TPGC), C11ORF49/CSTPP1, that regulates cytoskeletal organization, nuclear shape, and cilium disassembly. Mechanistically, loss of C11ORF49/CSTPP1 impacts the assembly and stability of the TPGC, which modulates long-chain polyglutamylation levels on microtubules (MTs) and thereby balances the binding of MT-associated proteins and actin nucleators. As a result, loss of TPGC leads to aberrant, enhanced assembly of MTs that penetrate the nucleus, which in turn leads to defects in nuclear shape, and disorganization of cytoplasmic actin that disrupts the YAP/TAZ pathway and cilium disassembly. Further, we showed that C11ORF49/CSTPP1-TPGC plays mechanistically distinct roles in the regulation of nuclear shape and cilium disassembly. Remarkably, disruption of C11ORF49/CSTPP1-TPGC also leads to developmental defects in vivo. Our findings point to an unanticipated nexus that links tubulin polyglutamylation with nuclear shape and ciliogenesis.

A non-canonical function for Centromere-associated protein-E controls centrosome integrity and orientation of cell division.

Centromere-associated protein-E (CENP-E) is a kinesin motor localizing at kinetochores. Although its mitotic functions have been well studied, it has been challenging to investigate direct consequences of CENP-E removal using conventional methods because CENP-E depletion resulted in mitotic arrest. In this study, we harnessed an auxin-inducible degron system to achieve acute degradation of CENP-E. We revealed a kinetochore-independent role for CENP-E that removes pericentriolar material 1 (PCM1) from centrosomes in late S/early G2 phase. After acute loss of CENP-E, centrosomal Polo-like kinase 1 (Plk1) localization is abrogated through accumulation of PCM1, resulting in aberrant phosphorylation and destabilization of centrosomes, which triggers shortened astral microtubules and oblique cell divisions. Furthermore, we also observed centrosome and cell division defects in cells from a microcephaly patient with mutations in CENPE. Orientation of cell division is deregulated in some microcephalic patients, and our unanticipated findings provide additional insights into how microcephaly can result from centrosomal defects.

Whole-genome screen identifies diverse pathways that negatively regulate ciliogenesis.

We performed a high-throughput whole-genome RNAi screen to identify novel inhibitors of ciliogenesis in normal and basal breast cancer cells. Our screen uncovered a previously undisclosed, extensive network of genes linking integrin signaling and cellular adhesion to the extracellular matrix (ECM) with inhibition of ciliation in both normal and cancer cells. Surprisingly, a cohort of genes encoding ECM proteins was also identified. We characterized several ciliation inhibitory genes and showed that their silencing was accompanied by altered cytoskeletal organization and induction of ciliation, which restricts cell growth and migration in normal and breast cancer cells. Conversely, supplying an integrin ligand, vitronectin, to the ECM rescued the enhanced ciliation observed on silencing this gene. Aberrant ciliation could also be suppressed through hyperactivation of the YAP/TAZ pathway, indicating a potential mechanistic basis for our findings. Our findings suggest an unanticipated reciprocal relationship between ciliation and cellular adhesion to the ECM and provide a resource that could vastly expand our understanding of controls involving "outside-in" and "inside-out" signaling that restrain cilium assembly.

Inner lumen proteins stabilize doublet microtubules in cilia and flagella.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

Assessment of the flagellar redox potential in Chlamydomonas reinhardtii using a redox-sensitive fluorescent protein, Oba-Qc.

Cellular reducing-oxidizing (redox) potential is mainly determined by the concentration ratio between reduced and oxidized glutathiones. It is normally kept at a moderately reduced state but affected to some extent by metabolic activities such as respiration and/or photosynthesis. Changes in redox potential induce many cellular activities collectively called redox responses. For an understanding of the dynamics of the cellular redox responses, redox potential must be accurately assessed in vivo. In this study, we developed a method to measure the in vivo redox potential in the green alga Chlamydomonas reinhardtii, using Oba-Qc, a recently developed redox-monitoring protein. Taking advantage of the periodic flagellar assembly, we introduced Oba-Qc molecules into the flagella at a constant density. Fluorescence signals from flagella in live cells, calibrated against the fluorescence from the samples in buffers of known redox potentials, determined the redox potential to be ∼-250 mV in the light and ∼-280 mV in the dark. Introduction of a sensor protein fused with a structural protein that assembles at a constant density will be also applicable for measurements of various kinds cellular signals in flagella.

Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms.

Cytoplasmic assembly of ciliary dyneins, a process known as preassembly, requires numerous non-dynein proteins, but the identities and functions of these proteins are not fully elucidated. Here, we show that the classical Chlamydomonas motility mutant pf23 is defective in the Chlamydomonas homolog of DYX1C1. The pf23 mutant has a 494 bp deletion in the DYX1C1 gene and expresses a shorter DYX1C1 protein in the cytoplasm. Structural analyses, using cryo-ET, reveal that pf23 axonemes lack most of the inner dynein arms. Spectral counting confirms that DYX1C1 is essential for the assembly of the majority of ciliary inner dynein arms (IDA) as well as a fraction of the outer dynein arms (ODA). A C-terminal truncation of DYX1C1 shows a reduction in a subset of these ciliary IDAs. Sucrose gradients of cytoplasmic extracts show that preassembled ciliary dyneins are reduced compared to wild-type, which suggests an important role in dynein complex stability. The role of PF23/DYX1C1 remains unknown, but we suggest that DYX1C1 could provide a scaffold for macromolecular assembly.

Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme.

Outer arm dynein (OAD) in cilia and flagella is bound to the outer doublet microtubules every 24 nm. Periodic binding of OADs at specific sites is important for efficient cilia/flagella beating; however, the molecular mechanism that specifies OAD arrangement remains elusive. Studies using the green alga Chlamydomonas reinhardtii have shown that the OAD-docking complex (ODA-DC), a heterotrimeric complex present at the OAD base, functions as the OAD docking site on the doublet. We find that the ODA-DC has an ellipsoidal shape ∼24 nm in length. In mutant axonemes that lack OAD but retain the ODA-DC, ODA-DC molecules are aligned in an end-to-end manner along the outer doublets. When flagella of a mutant lacking ODA-DCs are supplied with ODA-DCs upon gamete fusion, ODA-DC molecules first bind to the mutant axonemes in the proximal region, and the occupied region gradually extends toward the tip, followed by binding of OADs. This and other results indicate that a cooperative association of the ODA-DC underlies its function as the OAD-docking site and is the determinant of the 24-nm periodicity.

Protein-protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein.

Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association.