The life cycle of a primary cilium begins in quiescence and ends prior to mitosis. In quiescent cells, primary cilium insulates itself from contiguous dynamic membrane processes on the cell surface to function as a stable signaling apparatus. Here, we demonstrate that basal restriction of ciliary structure dynamics is established by cilia-enriched phosphoinositide 5-phosphatase, Inpp5e. Growth induction displaces ciliary Inpp5e and accumulates phosphatidylinositol 4,5-bisphosphate to distal cilia. This triggers otherwise forbidden actin polymerization in primary cilia, which excises cilia tips in a process we call cilia decapitation. Whilst cilia disassembly is traditionally thought to occur solely through resorption, we show that an acute loss of IFT-B through cilia decapitation precedes resorption. Finally, we propose that cilia decapitation induces mitogenic signaling and constitutes a molecular link between the cilia life cycle and cell-division cycle. This newly defined ciliary mechanism may find significance in cell proliferation control during normal development and cancer.

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Here, we show that cofilin-phosphatase activity of SSH1L increases ∼10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. We also provide evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1β induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin–rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1β and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm.

The precise alignment of chromosomes on the metaphase plate prior to the onset of anaphase is essential for ensuring equal segregation of sister chromatids into two daughter cells, and defects in this process potentially cause chromosome instability and tumor progression [1-3]. NDR1 is an evolutionarily conserved serine/threonine kinase whose activity is regulated by MST kinases, Furry (Fry), and MOB [4]. Although the NDR1 signaling pathway is implicated in cell division and morphogenesis in yeast and invertebrates [4-16], the mechanisms of NDR1 activation and the functional significance of the NDR1 pathway in mammalian cells are largely unknown. Here, we show that NDR1 is required for accurate chromosome alignment at metaphase in HeLa cells; depletion of NDR1, Fry, or MST2 caused mitotic chromosome misalignment. Chromosome misalignment in MST2-depleted cells was corrected by expression of active NDR1. The kinase activity of NDR1 increased in early mitotic phase and was dependent on Fry and MST2. We also provide evidence that Fry binds to microtubules, localizes on the spindle, acts as a scaffold that binds to both NDR1 and MOB2, and synergistically activates NDR1 with MOB2. Our findings suggest that MST2-, Fry-, and MOB2-mediated activation of NDR1 is crucial for the fidelity of mitotic chromosome alignment in mammalian cells.

Visualization of signal transduction within live primary cilia constitutes a technical challenge due to its sub-micron dimensions and close proximity to the cell body. Using a genetically encoded calcium indicator targeted to primary cilia we visualized calcium signaling in cilia of mouse fibroblasts and kidney cells upon chemical or mechanical stimulation with high specificity, sensitivity and wide dynamic range.

Mps one binder 1a (MOB1A) and MOB1B are key components of the Hippo signaling pathway and are mutated or inactivated in many human cancers. Here we show that intact Mob1a or Mob1b is essential for murine embryogenesis and that loss of the remaining WT Mob1 allele in Mob1a Δ/Δ 1b tr/+ or Mob1a Δ/+ 1b tr/tr mice results in tumor development. Because most of these cancers resembled trichilemmal carcinomas, we generated doublemutant mice bearing tamoxifen-inducible, keratinocyte-specific homozygous-null mutations of Mob1a and Mob1b (kDKO mice). kDKO mice showed hyperplastic keratinocyte progenitors and defective keratinocyte terminal differentiation and soon died of malnutrition. kDKO keratinocytes exhibited hyperproliferation, apoptotic resistance, impaired contact inhibition, enhanced progenitor self renewal, and increased centrosomes. Examination of Hippo pathway signaling in kDKO keratinocytes revealed that loss of Mob1a/b altered the activities of the downstream Hippo mediators LATS and YAP1. Similarly, YAP1 was activated in some human trichilemmal carcinomas, and some of these also exhibited MOB1A/1B inactivation. Our results clearly demonstrate that MOB1A and MOB1B have overlapping functions in skin homeostasis, and exert their roles as tumor suppressors by regulating downstream elements of the Hippo pathway.

tional experimentation following the publication of the paper has raised an issue with the results reported in Figure S2. The paper reported that disassembly of primary cilia can occur through ''decapitation'' of ciliary tips to release vesicles containing ciliary material. The phosphoinositide 5-phosphatase Inpp5e restricts the process. In Figure S2, the authors presented data supporting this role, showing that increased expression of Inpp5e promoted the suppressive effect and that the enzyme's catalytic activity was required. After publication, the authors discovered errors in the plasmids encoding 5HT 6-YFP-Inpp5e(WT)and 5HT 6-YFP-Inpp5e(PD, phosphatase dead), arising from the published DNA sequence of the parent plasmid, that resulted in expression plasmids with a frameshift between YFP and Inpp5e. They have repeated the same experiments with corrected constructs (available through Addgene-#96808, 96809), and obtained results consistent with the conclusions originally presented. With this note, we and the authors would like to alert the community and direct readers to additional data related to Figure S2, which have been deposited in Mendeley Data and can be accessed at https://data.mendeley.com/datasets/5vnt9wsbwf/1. The remaining findings in the paper are unchanged, and the new data do not alter the main conclusions of the paper. Additionally, the authors have informed us that they failed to list Sté phane Schurmans as an author despite a critical contribution to the work through generation of the Inpp5e-knockout MEFs that enabled collection of the data shown in Figures 1 and 3. To correct this oversight, we have added Sté phane Schurmans as an author. All co-authors have approved this addition and the corrected author list is shown below.

Primary cilia are antenna-like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi-derived vesicles to the pericentrosome, a process requiring Rab11-mediated recruitment of Rabin8, a GDP-GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser-272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8-/Rab11-containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP-bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho-mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore–microtubule attachment, chromosome alignment‐maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc‐finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1‐dependent phosphorylation at multiple sites during mitosis. CAMP‐depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K‐fibres to the tension exerted upon establishment of sister kinetochore bi‐orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C‐terminal region containing the zinc‐finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP‐E and CENP‐F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP‐E and CENP‐F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore–microtubule attachment during bi‐orientation.