Summary Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here, a global characterization of the neuronal palmitoyl-proteome identifies most of the known neuronal palmitoyl-proteins (PPs), 68 in total, plus over 200 new PP candidates, with additional testing confirming palmitoylation for 21 of these candidates. New PPs include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is a finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl) both with regard to localization and function: Cdc42-palm, concentrates in dendritic spines and plays a special role in inducing these post-synaptic structures. Finally, assessing palmitoylation dynamics in drug-induced activity paradigms finds rapidly induced changes both for Cdc42 as well as for other synaptic PPs, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.
Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging1-4. It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice5. Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.
Summary Background Condensin complexes organize chromosome structure and facilitate chromosome segregation. Higher eukaryotes have two complexes, condensin I and condensin II, each essential for chromosome segregation. The nematode Caenorhabditis elegans was considered an exception, because it has a mitotic condensin II complex but appeared to lack mitotic condensin I. Instead, its condensin I-like complex (here called condensin IDC) dampens gene expression along hermaphrodite X chromosomes during dosage compensation. Results Here we report the discovery of a third condensin complex, condensin I, in C. elegans. We identify new condensin subunits and show that each complex has a conserved five-subunit composition. Condensin I differs from condensin IDC by only a single subunit. Yet condensin I binds to autosomes and X chromosomes in both sexes to promote chromosome segregation, whereas condensin IDC binds specifically to X chromosomes in hermaphrodites to regulate transcript levels. Both condensin I and II promote chromosome segregation, but associate with different chromosomal regions during mitosis and meiosis. Unexpectedly, condensin I also localizes to regions of cohesion between meiotic chromosomes before their segregation. Conclusions We demonstrate that condensin subunits in C. elegans form three complexes, one that functions in dosage compensation and two that function in mitosis and meiosis. These results highlight how the duplication and divergence of condensin subunits during evolution may facilitate their adaptation to specialized chromosomal roles and illustrate the versatility of condensins to function in both gene regulation and chromosome segregation.
Summary The primary cilium is a microtubule-based organelle that senses extracellular signals as a cellular antenna [1]. Primary cilia are found on many types of cells in our body and play important roles in development and physiology. Defects of primary cilia cause a broad class of human genetic diseases called ciliopathies. To gain new insights into ciliary functions and better understand the molecular mechanisms underlying ciliopathies, it is of high importance to generate a catalog of primary cilia proteins. In this study, we isolated primary cilia from mouse kidney cells by using a calcium shock method and identified 195 candidate primary cilia proteins by MudPIT (multidimensional protein identification technology), protein correlation profiling, and subtractive proteomic analysis. Based on comparisons with other proteomic studies of cilia, around 75% of our candidate primary cilia proteins are shared components with motile or specialized sensory cilia. The remaining 25% of the candidate proteins are possible primary cilia specific proteins. These possible primary cilia specific proteins include Evc2, Inpp5e and Inversin, several of which have been linked to known ciliopathies. We have performed the first reported proteomic analysis of primary cilia from mammalian cells. These results provide new insights into primary cilia structure and function.
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