Project description:Inhibition of casein kinase 1 delta (CK1δ) blocks primary ciliogenesis in human telomerase reverse transcriptase immortalized retinal pigmented epithelial and mouse inner medullary collecting duct cells-3. Mouse embryonic fibroblasts (MEFs) and retinal cells from Csnk1d (CK1δ)-null mice also exhibit ciliogenesis defects. CK1δ catalytic activity and centrosomal localization signal (CLS) are required to rescue cilia formation in MEFs(Csnk1d null). Furthermore, expression of a truncated derivative containing the CLS displaces full-length CK1δ from the centrosome and decreases ciliary length in control MEFs, suggesting that centrosomal CK1δ has a role in ciliogenesis. CK1δ inhibition also alters pericentrosomal or ciliary distribution of several proteins involved in ciliary transport, including Ras-like in rat brain-11A, Ras-like in rat brain-8A, centrosomal protein of 290 kDa, pericentriolar material protein 1, and polycystin-2, as well as the Golgi distribution of its binding partner, A-kinase anchor protein 450 (AKAP450). As reported for AKAP450, CK1δ was required for microtubule nucleation at the Golgi and maintenance of Golgi integrity. Overexpression of an AKAP450 fragment containing the CK1δ-binding site inhibits Golgi-derived microtubule nucleation, Golgi distribution of intraflagellar transport protein 20 homologue, and ciliogenesis. Our results suggest that CK1δ mediates primary ciliogenesis by multiple mechanisms, one involving its centrosomal function and another dependent on its interaction with AKAP450 at the Golgi, where it is important for maintaining Golgi organization and polarized trafficking of multiple factors that mediate ciliary transport.
Project description:Activation of the cAMP-dependent protein kinase (PKA) is critical for both short- and long-term facilitation in Aplysia sensory neurons. There are two types of the kinase, I and II, differing in their regulatory (R) subunits. We cloned Aplysia RII; RI was cloned previously. Type I PKA is mostly soluble in the cell body whereas type II is enriched at nerve endings where it is bound to two prominent A kinase-anchoring-proteins (AKAPs). Disruption of the binding of RII to AKAPs by Ht31, an inhibitory peptide derived from a human thyroid AKAP, prevents both the short- and the long-term facilitation produced by serotonin (5-HT). During long-term facilitation, RII is transcriptionally upregulated; in contrast, the amount of RI subunits decreases, and previous studies have indicated that the decrease is through ubiquitin-proteosome-mediated proteolysis. Experiments with antisense oligonucleotides injected into the sensory neuron cell body show that the increase in RII protein is essential for the production of long-term facilitation. Using synaptosomes, we found that 5-HT treatment causes RII protein to increase at nerve endings. In addition, using reverse transcription-PCR, we found that RII mRNA is transported from the cell body to nerve terminals. Our results suggest that type I operates in the nucleus to maintain cAMP response element-binding protein-dependent gene expression, and type II PKA acts at sensory neuron synapses phosphorylating proteins to enhance release of neurotransmitter. Thus, the two types of the kinase have distinct but complementary functions in the production of facilitation at synapses of an identified neuron.
Project description:Almost all brain cells contain cilia, antennae-like microtubule-based organelles. Yet, the significance of cilia, once considered vestigial organelles, in the higher-order brain functions is unknown. Cilia act as a hub that senses and transduces environmental sensory stimuli to generate an appropriate cellular response. Similarly, the striatum, a brain structure enriched in cilia, functions as a hub that receives and integrates various types of environmental information to drive appropriate motor response. To understand cilia's role in the striatum functions, we used loxP/Cre technology to ablate cilia from the dorsal striatum of male mice and monitored the behavioral consequences. Our results revealed an essential role for striatal cilia in the acquisition and brief storage of information, including learning new motor skills, but not in long-term consolidation of information or maintaining habitual/learned motor skills. A fundamental aspect of all disrupted functions was the "time perception/judgment deficit." Furthermore, the observed behavioral deficits form a cluster pertaining to clinical manifestations overlapping across psychiatric disorders that involve the striatum functions and are known to exhibit timing deficits. Thus, striatal cilia may act as a calibrator of the timing functions of the basal ganglia-cortical circuit by maintaining proper timing perception. Our findings suggest that dysfunctional cilia may contribute to the pathophysiology of neuro-psychiatric disorders, as related to deficits in timing perception.
Project description:Activated Wnt/beta-catenin signalling is a characteristic of many cancers and drives cell-cycle progression. Here, we report a mechanism linking Wnt/beta-catenin signalling to centrosome separation. We show that conductin/axin2, a negative regulator of beta-catenin, localizes at the centrosomes by binding to the centriole-associated component C-Nap1. Knockout or knockdown of conductin leads to premature centrosome separation--that is, splitting--which is abolished by knockdown of beta-catenin. Conductin promotes phosphorylation of the amino-terminal serine (Ser 33/37) and threonine (Thr 41) residues of centrosome-associated beta-catenin. Beta-catenin mutated at these residues causes centrosomal splitting, whereas a phospho-mimicking mutant of beta-catenin does not. Importantly, beta-catenin-induced splitting is not inhibited by blocking beta-catenin-dependent transcription. Treatment with Wnts and inhibition of glycogen synthase kinase 3 block beta-catenin phosphorylation and induce centrosomal splitting. These data indicate that Wnt/beta-catenin signalling and conductin regulate centrosomal cohesion by altering the phosphorylation status of beta-catenin at the centrosomes.
Project description:The phosphoinositide 3-kinase (PI3K)/phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K/PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K/PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt(473) show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.
Project description:The centrosome linker serves to hold the duplicated centrosomes together until they separate in late G2/early mitosis. Precisely how the linker is assembled remains an open question. In this study, we identify Cep44 as a novel component of the linker in human cells. Cep44 localizes to the proximal end of centrioles, including mother and daughter centrioles, and its ablation leads to loss of centrosome cohesion. Cep44 does not impinge on the stability of C-Nap1 (also known as CEP250), LRRC45 or Cep215 (also known as CDK5RAP2), and vice versa, and these proteins are independently recruited to the centrosome. Rather, Cep44 associates with rootletin and regulates its stability and localization to the centrosome. Our findings reveal a role of the previously uncharacterized protein Cep44 for centrosome cohesion and linker assembly.
Project description:Asymmetric astral microtubule organization drives the polarized orientation of the S. cerevisiae mitotic spindle and primes the invariant inheritance of the old spindle pole body (SPB, the yeast centrosome) by the bud. This model has anticipated analogous centrosome asymmetries featured in self-renewing stem cell divisions. We previously implicated Spc72, the cytoplasmic receptor for the gamma-tubulin nucleation complex, as the most upstream determinant linking SPB age, functional asymmetry and fate. Here we used structured illumination microscopy and biochemical analysis to explore the asymmetric landscape of nucleation sites inherently built into the spindle pathway and under the control of cyclin-dependent kinase (CDK). We show that CDK enforces Spc72 asymmetric docking by phosphorylating Nud1/centriolin. Furthermore, CDK-imposed order in the construction of the new SPB promotes the correct balance of nucleation sites between the nuclear and cytoplasmic faces of the SPB. Together these contributions by CDK inherently link correct SPB morphogenesis, age and fate.
Project description:Centrosome amplification (CA) has been implicated in the progression of various cancer types. Although studies have shown that overexpression of PLK4 promotes CA, the effect of tumor microenvironment on polo-like kinase 4 (PLK4) regulation is understudied. The aim of this study was to examine the role of hypoxia in promoting CA via PLK4. We found that hypoxia induced CA via hypoxia-inducible factor-1α (HIF1α). We quantified the prevalence of CA in tumor cell lines and tissue sections from breast cancer, pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, and prostate cancer and found that CA was prevalent in cells with increased HIF1α levels under normoxic conditions. HIF1α levels were correlated with the extent of CA and PLK4 expression in clinical samples. We analyzed the correlation between PLK4 and HIF1A mRNA levels in The Cancer Genome Atlas (TCGA) datasets to evaluate the role of PLK4 and HIF1α in breast cancer and PDAC prognosis. High HIF1A and PLK4 levels in patients with breast cancer and PDAC were associated with poor overall survival. We confirmed PLK4 as a transcriptional target of HIF1α and demonstrated that in PLK4 knockdown cells, hypoxia-mimicking agents did not affect CA and expression of CA-associated proteins, underscoring the necessity of PLK4 in HIF1α-related CA. To further dissect the HIF1α-PLK4 interplay, we used HIF1α-deficient cells overexpressing PLK4 and showed a significant increase in CA compared with HIF1α-deficient cells harboring wild-type PLK4. These findings suggest that HIF1α induces CA by directly upregulating PLK4 and could help us risk-stratify patients and design new therapies for CA-rich cancers.ImplicationsHypoxia drives CA in cancer cells by regulating expression of PLK4, uncovering a novel HIF1α/PLK4 axis.
Project description:Chromosomal translocations observed in myeloproliferative neoplasms (MPNs) frequently fuse genes that encode centrosome proteins and tyrosine kinases. This causes constitutive activation of the kinase resulting in aberrant, proliferative signaling. The function of centrosome proteins in these fusions is not well understood. Among others, kinase centrosome localization and constitutive kinase dimerization are possible consequences of centrosome protein-kinase fusions. To test the relative contributions of localization and dimerization on kinase signaling, we targeted inducibly dimerizable FGFR1 to the centrosome and other subcellular locations and generated a mutant of the FOP-FGFR1 MPN fusion defective in centrosome localization. Expression in mammalian cells followed by western blot analysis revealed a significant decrease in kinase signaling upon loss of FOP-FGFR1 centrosome localization. Kinase dimerization alone resulted in phosphorylation of the FGFR1 signaling target PLCγ, however levels comparable to FOP-FGFR1 required subcellular targeting in addition to kinase dimerization. Expression of MPN fusion proteins also resulted in centrosome disruption in epithelial cells and transformed patient cells. Primary human MPN cells showed masses of modified tubulin that colocalized with centrin, Smoothened (Smo), IFT88, and Arl13b. This is distinct from acute myeloid leukemia (AML) cells, which are not associated with centrosome-kinase fusions and had normal centrosomes. Our results suggest that effective proliferative MPN signaling requires both subcellular localization and dimerization of MPN kinases, both of which may be provided by centrosome protein fusion partners. Furthermore, centrosome disruption may contribute to the MPN transformation phenotype.