Intraflagellar transport proteins are involved in thrombocyte filopodia formation and secretion.

Intraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to thrombocyte aggregation (TTA) in the plate tilt assay after thrombocyte activation by the following agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in thrombocyte function.

CDK5RAP2 regulates centriole engagement and cohesion in mice.

Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microcephaly in humans. Here we show that CDK5RAP2 loss of function in mice causes centriole amplification with a preponderance of single, unpaired centrioles and increased numbers of daughter-daughter centriole pairs. These results indicate that CDK5RAP2 is required to maintain centriole engagement and cohesion, thereby restricting centriole replication. Early in mitosis, amplified centrosomes assemble multipolar spindles in CDK5RAP2 mutant cells. Moreover, both mother and daughter centrioles are amplified and the excess mother centrioles template multiple primary cilia in CDK5RAP2 mutant cells.

Cilia proteins control cerebellar morphogenesis by promoting expansion of the granule progenitor pool.

Although human congenital cerebellar malformations are common, their molecular and developmental basis is still poorly understood. Recently, cilia-related gene deficiencies have been implicated in several congenital disorders that exhibit cerebellar abnormalities such as Joubert syndrome, Meckel-Gruber syndrome, Bardet-Biedl syndrome, and Orofaciodigital syndrome. The association of cilia gene mutations with these syndromes suggests that cilia may be important for cerebellar development, but the nature of cilia involvement has not been elucidated. To assess the importance of cilia-related proteins during cerebellar development, we studied the effects of CNS-specific inactivation of two mouse genes whose protein products are critical for cilia formation and maintenance, IFT88, (also known as polaris or Tg737), which encodes intraflagellar transport 88 homolog, and Kif3a, which encodes kinesin family member 3a. We showed that loss of either of these genes caused severe cerebellar hypoplasia and foliation abnormalities, primarily attributable to a failure of expansion of the neonatal granule cell progenitor population. In addition, granule cell progenitor proliferation was sensitive to partial loss of IFT function in a hypomorphic mutant of IFT88 (IFT88(orpk)), an effect that was modified by genetic background. IFT88 and Kif3a were not required for the specification and differentiation of most other cerebellar cell types, including Purkinje cells. Together, our observations constitute the first demonstration that cilia proteins are essential for normal cerebellar development and suggest that granule cell proliferation defects may be central to the cerebellar pathology in human cilia-related disorders.

Peripheral inflammation increases phosphoinositide activity in the rat dorsal horn.

Persistent pain leads to changes in the spinal cord that contribute to hyperalgesia and allodynia. The effort to characterize these changes has focused on neurotransmitters and receptors, while relatively little is known about pain-associated modulation of second-messenger responses. Nearly all neurotransmitters can activate the phosphoinositide (PI) second-messenger system which has been investigated using a method that localizes membrane-bound [(3)H]CDP-diacylglycerol (DAG) produced from the precursor [(3)H]cytidine [Science 249 (1990) 802]. The present study applied this method in spinal cord slices from rats injected with complete Freund's adjuvant in one hindpaw and from uninflamed control rats. Two days after the injection, slices were removed and maintained in vitro for pharmacological testing. Some slices were exposed to the acetylcholine agonist carbachol which is antinociceptive in the spinal cord. Inflammation resulted in increased baseline, unstimulated [(3)H]CDP-DAG accumulation, especially in superficial dorsal horn layers, as well as enhanced carbachol-stimulated labeling. These results suggest that persistent pain leads to neurochemical changes within the spinal cord that could potentially enhance responses to a spectrum of pain-modulating transmitters.

Neuronal primary cilia: a review.

Primary cilia in neurons have often been regarded as rare, vestigial curiosities. However, neuronal cilia are now gaining recognition as ubiquitous organelles in the mammalian brain, raising speculation about what their functions may be. They might have some features tailored for the nervous system and others that serve needs shared by a spectrum of other cell types. Here we review clues from the literature and present new data supporting several possibilities for the significance of neuronal cilia. Our immunocytochemical results show regional heterogeneity in neuronal cilia. Brain regions nearer to the cerebral ventricles had longer cilia, suggesting that they might sense chemicals such as peptides, originating from cerebrospinal fluid. In mutant Tg737(orpk)mice, most brain regions appeared to be missing cilia. The importance of intraflagellar transport proteins establishes a functional link between neuronal cilia and other primary cilia.