Despite the strong evidence linking the aggregation of the Huntingtin protein (Htt) to the pathogenesis of Huntington's disease (HD), the mechanisms underlying Htt aggregation and neurodegeneration ...remain poorly understood. Herein, we investigated the ultrastructural properties and protein composition of Htt cytoplasmic and nuclear inclusions in mammalian cells and primary neurons overexpressing mutant exon1 of the Htt protein. Our findings provide unique insight into the ultrastructural properties of cytoplasmic and nuclear Htt inclusions and their mechanisms of formation. We show that Htt inclusion formation and maturation are complex processes that, although initially driven by polyQ-dependent Htt aggregation, also involve the polyQ and PRD domain-dependent sequestration of lipids and cytoplasmic and cytoskeletal proteins related to HD dysregulated pathways; the recruitment and accumulation of remodeled or dysfunctional membranous organelles, and the impairment of the protein quality control and degradation machinery. We also show that nuclear and cytoplasmic Htt inclusions exhibit distinct biochemical compositions and ultrastructural properties, suggesting different mechanisms of aggregation and toxicity.
Mutations in the MFN2 gene encoding Mitofusin 2 lead to the development of Charcot–Marie–Tooth type 2A (CMT2A), a dominant axonal form of peripheral neuropathy. Mitofusin 2 is localized at both the ...outer membrane of mitochondria and the endoplasmic reticulum and is particularly enriched at specialized contact regions known as mitochondria-associated membranes (MAM). We observed that expression of MFN2R94Q induces distal axonal degeneration in the absence of overt neuronal death. The presence of mutant protein leads to reduction in endoplasmic reticulum and mitochondria contacts in CMT2A patient-derived fibroblasts, in primary neurons and in vivo, in motoneurons of a mouse model of CMT2A. These changes are concomitant with endoplasmic reticulum stress, calcium handling defects, and changes in the geometry and axonal transport of mitochondria. Importantly, pharmacological treatments reinforcing endoplasmic reticulum–mitochondria cross-talk, or reducing endoplasmic reticulum stress, restore the mitochondria morphology and prevent axonal degeneration. These results highlight defects in MAM as a cellular mechanism contributing to CMT2A pathology mediated by mutated MFN2.
Parkinson’s disease (PD) is characterized by the accumulation of misfolded and aggregated α-synuclein (α-syn) into intraneuronal inclusions named Lewy bodies (LBs). Although it is widely believed ...that α-syn plays a central role in the pathogenesis of PD, the processes that govern α-syn fibrillization and LB formation remain poorly understood. In this work, we sought to dissect the spatiotemporal events involved in the biogenesis of the LBs at the genetic, molecular, biochemical, structural, and cellular levels. Toward this goal, we further developed a seeding-based model of α-syn fibrillization to generate a neuronal model that reproduces the key events leading to LB formation, including seeding, fibrillization, and the formation of inclusions that recapitulate many of the biochemical, structural, and organizational features of bona fide LBs. Using an integrative omics, biochemical and imaging approach, we dissected the molecular events associated with the different stages of LB formation and their contribution to neuronal dysfunction and degeneration. In addition, we demonstrate that LB formation involves a complex interplay between α-syn fibrillization, posttranslational modifications, and interactions between α-syn aggregates and membranous organelles, including mitochondria, the autophagosome, and endolysosome. Finally, we show that the process of LB formation, rather than simply fibril formation, is one of the major drivers of neurodegeneration through disruption of cellular functions and inducing mitochondria damage and deficits, and synaptic dysfunctions. We believe that this model represents a powerful platform to further investigate the mechanisms of LB formation and clearance and to screen and evaluate therapeutics targeting α-syn aggregation and LB formation.
In most metazoans, centrioles are lost during oogenesis, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is ...dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in Caenorhabditis elegans. Centriole elimination begins with loss of the so‐called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule‐ and dynein‐dependent manner to the plasma membrane. Our analysis indicates that neither Polo‐like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS‐1 normally departs initially from the organelle, which loses integrity earlier in sas‐1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.
Synopsis
Paternal contribution of centrioles ensures centriole homeostasis in zygotes, but how maternal centrioles are lost in metazoans is unclear. Detailed ultrastructural analyses of centriole elimination during worm oogenesis show that departure of the central tube protein SAS‐1 marks the onset of organelle disassembly.
Oogenesis centriole elimination in C. elegans occurs in two steps.
Loss of ultrastructural integrity precedes clearance of centriolar protein cluster.
Central tube removal and organelle widening mark the onset of centriole elimination.
The central tube protein SAS‐1 is required for centriole stability during oogenesis.
A combination of ultastructural analyses shows that centrioles are eliminated via two distinct steps during oogenesis in worms, marked by central tube removal and organelle widening.
Mitochondria contain the genetic information and expression machinery to produce essential respiratory chain proteins. Within the mitochondrial matrix, newly synthesized RNA, RNA processing proteins ...and mitoribosome assembly factors form punctate sub-compartments referred to as mitochondrial RNA granules (MRGs)
. Despite their proposed importance in regulating gene expression, the structural and dynamic properties of MRGs remain largely unknown. We investigated the internal architecture of MRGs using fluorescence super-resolution localization microscopy and correlative electron microscopy, and found that the MRG ultrastructure consists of compacted RNA embedded within a protein cloud. Using live-cell super-resolution structured illumination microscopy and fluorescence recovery after photobleaching, we reveal that MRGs rapidly exchange components and can undergo fusion, characteristic properties of fluid condensates
. Furthermore, MRGs associate with the inner mitochondrial membrane and their fusion coincides with mitochondrial remodelling. Inhibition of mitochondrial fission or fusion leads to an aberrant accumulation of MRGs into concentrated pockets, where they remain as distinct individual units despite their close apposition. Together, our findings reveal that MRGs are nanoscale fluid compartments, which are dispersed along mitochondria via membrane dynamics.
Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of ...cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and “caps” microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control.
•CPAP's PN2-3 domain binds to an exposed site on β-tubulin at microtubule plus ends•CPAP tracks and caps microtubule plus ends in vitro•CPAP dampens microtubule growth in vitro•The capping function of CPAP limits centriolar microtubule growth in human cells
The mechanisms ensuring the extremely slow growth of centriolar microtubules remain elusive. Sharma, Aher, Dynes et al. demonstrate that human CPAP acts as a molecular lid that caps microtubule plus ends and dampens their elongation, thus contributing to centriole length control by ensuring slow processive assembly of centriolar microtubules.
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