Faculty Bibliography

Many proteins are known to promote ciliogenesis, but mechanisms that promote primary cilia disassembly before mitosis are largely unknown. Here we identify a mechanism that favours cilium disassembly and maintains the disassembled state. We show that co-localization of the S/G2 phase kinase, Nek2 and Kif24 triggers Kif24 phosphorylation, inhibiting cilia formation. We show that Kif24, a microtubule depolymerizing kinesin, is phosphorylated by Nek2, which stimulates its activity and prevents the outgrowth of cilia in proliferating cells, independent of Aurora A and HDAC6. Our data also suggest that cilium assembly and disassembly are in dynamic equilibrium, but Nek2 and Kif24 can shift the balance toward disassembly. Further, Nek2 and Kif24 are overexpressed in breast cancer cells, and ablation of these proteins restores ciliation in these cells, thereby reducing proliferation. Thus, Kif24 is a physiological substrate of Nek2, which regulates cilia disassembly through a concerted mechanism involving Kif24-mediated microtubule depolymerization.

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.

The primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly. Here, we report that kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity at the mother centriole to disassemble the primary cilium in a growth-signal-dependent manner. KIF2A-deficient hTERT-RPE1 cells showed the impairment of primary cilia disassembly following growth stimulation. It was also found that the PLK1-KIF2A pathway is constitutively active in cells from patients with premature chromatid separation (PCS) syndrome and is responsible for defective ciliogenesis in this syndrome. These findings provide insights into the roles of the PLK1-KIF2A pathway in physiological cilia disassembly and cilia-associated disorders.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Autophagy is the primary catabolic process triggered in response to starvation. Although autophagic regulation within the cytosolic compartment is well established, it is becoming clear that nuclear events also regulate the induction or repression of autophagy. Nevertheless, a thorough understanding of the mechanisms by which sequence-specific transcription factors modulate expression of genes required for autophagy is lacking. Here, we identify Foxk proteins (Foxk1 and Foxk2) as transcriptional repressors of autophagy in muscle cells and fibroblasts. Interestingly, Foxk1/2 serve to counter-balance another forkhead transcription factor, Foxo3, which induces an overlapping set of autophagic and atrophic targets in muscle. Foxk1/2 specifically recruits Sin3A-HDAC complexes to restrict acetylation of histone H4 and expression of critical autophagy genes. Remarkably, mTOR promotes the transcriptional activity of Foxk1 by facilitating nuclear entry to specifically limit basal levels of autophagy in nutrient-rich conditions. Our study highlights an ancient, conserved mechanism whereby nutritional status is interpreted by mTOR to restrict autophagy by repressing essential autophagy genes through Foxk-Sin3-mediated transcriptional control.

The primary cilium acts as a cellular antenna, transducing diverse signaling pathways, and recent evidence suggests that primary cilia are important in development and cancer. However, a role for cilia in normal muscle development and rhabdomyosarcoma (RMS) has not been explored. Here we implicate primary cilia in proliferation, hedgehog (Hh) signaling, and differentiation of skeletal muscle cells. Cilia and Hh signaling are highly dynamic during the differentiation of myoblasts. We show that cilia are assembled during the initial stages of myogenic differentiation but disappear as cells progress through myogenesis, concomitant with the destruction of proteins critical for cilia assembly and shortly after the Hh effector, Gli3, leaves the cilium. Importantly, we show that ablation of primary cilia strongly suppresses Hh signaling and myogenic differentiation while enhancing proliferation. Interestingly, our data further indicate that both cilia assembly and Hh signaling are deregulated in RMS, and cilia respond to Hh ligand in certain subsets of RMS cells but not others. Together, these findings provide evidence for an essential role for both primary cilia assembly and disassembly in the control of Hh signaling and early differentiation in muscle cells. We suggest that the temporally orchestrated destruction of centrosomal and ciliary proteins is a necessary antecedent for removal of the primary cilium and cessation of Hh signaling during myogenic differentiation. Additionally, our results further stratify RMS populations and highlight cilia assembly and disassembly as potential RMS drug targets.

Monomethylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we uncover roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes monomethylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Monomethylation-mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human embryonic stem cells (ESCs). On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 monomethylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.

We have identified Talpid3/KIAA0586 as a component of a CP110-containing protein complex important for centrosome and cilia function. Talpid3 assembles a ring-like structure at the extreme distal end of centrioles. Ablation of Talpid3 resulted in an aberrant distribution of centriolar satellites involved in protein trafficking to centrosomes as well as cilia assembly defects, reminiscent of loss of Cep290, another CP110-associated protein. Talpid3 depletion also led to mislocalization of Rab8a, a small GTPase thought to be essential for ciliary vesicle formation. Expression of activated Rab8a suppressed cilia assembly defects provoked by Talpid3 depletion, suggesting that Talpid3 affects cilia formation through Rab8a recruitment and/or activation. Remarkably, ultrastructural analyses showed that Talpid3 is required for centriolar satellite dispersal, which precedes the formation of mature ciliary vesicles, a process requiring Cep290. These studies suggest that Talpid3 and Cep290 play overlapping and distinct roles in ciliary vesicle formation through regulation of centriolar satellite accretion and Rab8a.

Cilia are hair-like protrusions found at the surface of most eukaryotic cells. They can be divided into two types, motile and non-motile. Motile cilia are found in a restricted number of cell types, are generally present in large numbers, and beat in a coordinated fashion to generate fluid flow or locomotion. Non-motile or primary cilia, on the other hand, are detected in many different cell types, appear once per cell, and primarily function to transmit signals from the extracellular milieu to the cell nucleus. Defects in cilia formation, function, or maintenance are known to cause a bewildering set of human diseases, or ciliopathies, typified by retinal degeneration, renal failure and cystic kidneys, obesity, liver dysfunction, and neurological disorders. A common denominator between motile and primary cilia is their structural similarity, as both types of cilia are composed of an axoneme, the ciliary backbone that is made up of microtubules emanating from a mother centriole/basal body anchored to the cell membrane, surrounded by a ciliary membrane continuous with the plasma membrane. This structural similarity is indicative of a universal mechanism of cilia assembly involving a common set of molecular players and a sophisticated, highly regulated series of molecular events. In this review, we will mainly focus on recent advances in our understanding of the regulatory mechanisms underlying cilia assembly, with special attention paid to the centriolar protein, CP110, its interacting partner Cep290, and the various downstream molecular players and events leading to intraflagellar transport (IFT), a process that mediates the bidirectional movement of protein cargos along the axoneme and that is essential for cilia formation and maintenance.