The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo–electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

The charged multivesicular body proteins (Chmp1-7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause frontotemporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, decreased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b-containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines.

Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity.

Alix (ALG-2 interacting protein X) drives deformation and fission of endosomal and cell surface membranes and thereby intervenes in diverse biological processes including cell proliferation and apoptosis. Using embryonic fibroblasts of Alix knock-out mice, we recently demonstrated that Alix is required for clathrin-independent endocytosis. Here we show that mice lacking Alix suffer from severe reduction in the volume of the brain which affects equally all regions examined. The cerebral cortex of adult animals shows normal layering but is reduced in both medio-lateral length and thickness. Alix controls brain size by regulating its expansion during two distinct developmental stages. Indeed, embryonic surface expansion of the Alix ko cortex is reduced because of the loss of neural progenitors during a transient phase of apoptosis occurring between E11.5 and E12.5. Subsequent development of the Alix ko cortex occurs normally until birth, when Alix is again required for the post-natal radial expansion of the cortex through its capacity to allow proper neurite outgrowth. The need of Alix for both survival of neural progenitor cells and neurite outgrowth is correlated with its role in clathrin-independent endocytosis in neural progenitors and at growth cones. Thus Alix-dependent, clathrin independent endocytosis is essential for controlling brain size.

The molecular mechanisms and the biological functions of clathrin independent endocytosis (CIE) remain largely elusive. Alix (ALG-2 interacting protein X), has been assigned roles in membrane deformation and fission both in endosomes and at the plasma membrane. Using Alix ko cells, we show for the first time that Alix regulates fluid phase endocytosis and internalization of cargoes entering cells via CIE, but has no apparent effect on clathrin mediated endocytosis or downstream endosomal trafficking. We show that Alix acts with endophilin-A to promote CIE of cholera toxin and to regulate cell migration. We also found that Alix is required for fast endocytosis and downstream signaling of the interleukin-2 receptor giving a first indication that CIE is necessary for activation of at least some surface receptors. In addition to characterizing a new function for Alix, our results highlight Alix ko cells as a unique tool to unravel the biological consequences of CIE.

Cryofixation has proven to be the gold standard for efficient preservation of native cell ultrastructure compared to chemical fixation, but this approach is not widely used in fluorescence microscopy owing to implementation challenges. Here, we develop Cryo-ExM, a method that preserves native cellular organization by coupling cryofixation with expansion microscopy. This method bypasses artifacts associated with chemical fixation and its simplicity will contribute to its widespread use in super-resolution microscopy.

No abstract

Over the course of evolution, the centrosome function has been conserved in most eukaryotes, but its core architecture has evolved differently in some clades, with the presence of centrioles in humans and a spindle pole body (SPB) in yeast. Similarly, the composition of these two core elements has diverged, with the exception of Centrin and SFI1, which form a complex in yeast to initiate SPB duplication. However, it remains unclear whether this complex exists at centrioles and whether its function has been conserved. Here, using expansion microscopy, we demonstrate that human SFI1 is a centriolar protein that associates with a pool of Centrin at the distal end of the centriole. We also find that both proteins are recruited early during procentriole assembly and that depletion of SFI1 results in the loss of the distal pool of Centrin, without altering centriole duplication. Instead, we show that SFI1/Centrin complex is essential for centriolar architecture, CEP164 distribution, and CP110 removal during ciliogenesis. Together, our work reveals a conserved SFI1/Centrin module displaying divergent functions between mammals and yeast.