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Distal appendages (DAPs) are nanoscale, pinwheel-like structures protruding from the distal end of the centriole that mediate membrane docking during ciliogenesis, marking the cilia base around the ciliary gate. Here we determine a super-resolved multiplex of 16 centriole-distal-end components. Surprisingly, rather than pinwheels, intact DAPs exhibit a cone-shaped architecture with components filling the space between each pinwheel blade, a new structural element we term the distal appendage matrix (DAM). Specifically, CEP83, CEP89, SCLT1, and CEP164 form the backbone of pinwheel blades, with CEP83 confined at the root and CEP164 extending to the tip near the membrane-docking site. By contrast, FBF1 marks the distal end of the DAM near the ciliary membrane. Strikingly, unlike CEP164, which is essential for ciliogenesis, FBF1 is required for ciliary gating of transmembrane proteins, revealing DAPs as an essential component of the ciliary gate. Our findings redefine both the structure and function of DAPs. Distal appendages (DAPs) at the cilia base mediate membrane docking during ciliogenesis. Here the authors use super-resolution microscopy to map 16 centriole distal end components, revealing the structure of the backbone of the DAP, as well as a previously undescribed distal appendage matrix.

Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11–Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11–Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth. Westlake and colleagues discover that membrane shaping EHD proteins participate in ciliogenesis by taking part in ciliary vesicle formation and transition zone protein recruitment.

Clinical and experimental observations indicate a role for VEGF secreted by the retinal pigment epithelium (RPE) in the maintenance of the choriocapillaris (CC). VEGF in mice is produced as three isoforms, VEGF120, VEGF164, and VEGF188, that differ in their ability to bind heparan sulfate proteoglycan. RPE normally produces the more soluble isoforms, VEGF120 and VEGF164, but virtually no VEGF188, reflecting the fact that molecules secreted by the RPE must diffuse across Bruch's membrane (BrM) to reach the choriocapillaris. To determine the role of RPE-derived soluble VEGF on the choriocapillaris survival, we used mice that produce only VEGF188. VEGF188/188 mice exhibited normal choriocapillaris development. However, beginning at 7 months of age, we observed a progressive degeneration characterized by choriocapillaris atrophy, RPE and BrM abnormalities, culminating in areas of RPE loss and dramatic choroidal remodeling. Increased photoreceptor apoptosis in aged VEGF188/188 mice led to a decline in visual acuity as detected by electroretinogram (ERG). These changes are reminiscent of geographic atrophy (GA) and point to a role for RPE-derived VEGF in the maintenance of the choriocapillaris.

Vaginal atrophy occurring during menopause is closely related to the dramatic decrease in ovarian estrogens due to the loss of follicular activity. Particularly, significant changes occur in the structure of the vaginal mucosa, with consequent impairment of many physiological functions. In this study, carried out on bioptic vaginal mucosa samples from postmenopausal, nonestrogenized women, we present microscopic and ultrastructural modifications of vaginal mucosa following fractional carbon dioxide (CO 2 ) laser treatment. We observed the restoration of the vaginal thick squamous stratified epithelium with a significant storage of glycogen in the epithelial cells and a high degree of glycogen-rich shedding cells at the epithelial surface. Moreover, in the connective tissue constituting the lamina propria, active fibroblasts synthesized new components of the extracellular matrix including collagen and ground substance (extrafibrillar matrix) molecules. Differently from atrophic mucosa, newly-formed papillae of connective tissue indented in the epithelium and typical blood capillaries penetrating inside the papillae, were also observed. Our morphological findings support the effectiveness of fractional CO 2 laser application for the restoration of vaginal mucosa structure and related physiological trophism. These findings clearly coupled with striking clinical relief from symptoms suffered by the patients before treatment.

Multiwalled carbon nanotubes (MWCNTs) have the potential for widespread applications in engineering and materials science. However, because of their needle-like shape and high durability, concerns have been raised that MWCNTs may induce asbestos-like pathogenicity. Although recent studies have demonstrated that MWCNTs induce various types of reactivities, the physicochemical features of MWCNTs that determine their cytotoxicity and carcinogenicity in mesothelial cells remain unclear. Here, we showed that the deleterious effects of nonfunctionalized MWCNTs on human mesothelial cells were associated with their diameter-dependent piercing of the cell membrane. Thin MWCNTs (diameter ∼ 50 nm) with high crystallinity showed mesothelial cell membrane piercing and cytotoxicity in vitro and subsequent inflammogenicity and mesotheliomagenicity in vivo. In contrast, thick (diameter ∼ 150 nm) or tangled (diameter ∼ 2–20 nm) MWCNTs were less toxic, inflammogenic, and carcinogenic. Thin and thick MWCNTs similarly affected macrophages. Mesotheliomas induced by MWCNTs shared homozygous deletion of Cdkn2a/2b tumor suppressor genes, similar to mesotheliomas induced by asbestos. Thus, we propose that different degrees of direct mesothelial injury by thin and thick MWCNTs are responsible for the extent of inflammogenicity and carcinogenicity. This work suggests that control of the diameter of MWCNTs could reduce the potential hazard to human health.

Oral delivery of protein drugs is considered a life-changing solution for patients who require regular needle injections. However, clinical translation of oral protein formulations has been hampered by inefficient penetration of drugs through the intestinal mucus and epithelial cell layer, leading to low absorption and bioavailability, and safety concerns owing to tight junction openings. Here we report a zwitterionic micelle platform featuring a virus-mimetic zwitterionic surface, a betaine side chain and an ultralow critical micelle concentration, enabling drug penetration through the mucus and efficient transporter-mediated epithelial absorption without the need for tight junction opening. This micelle platform was used to fabricate a prototype oral insulin formulation by encapsulating a freeze-dried powder of zwitterionic micelle insulin into an enteric-coated capsule. The biocompatible oral insulin formulation shows a high oral bioavailability of >40%, offers the possibility to fine tune insulin acting profiles and provides long-term safety, enabling the oral delivery of protein drugs.A zwitterionic micelle platform enables the oral delivery and high bioavailability of insulin by transporting the protein across mucus and epithelial barriers in the gut without opening tight junctions.

Sodium iodate (SI) is a widely used oxidant for generating retinal degeneration models by inducing the death of retinal pigment epithelium (RPE) cells. However, the mechanism of RPE cell death induced by SI remains unclear. In this study, we investigated the necrotic features of cultured human retinal pigment epithelium (ARPE-19) cells treated with SI and found that apoptosis or necroptosis was not the major death pathway. Instead, the death process was accompanied by significant elevation of intracellular labile iron level, ROS, and lipid peroxides which recapitulated the key features of ferroptosis. Ferroptosis inhibitors deferoxamine mesylate (DFO) and ferrostatin-1(Fer-1) partially prevented SI-induced cell death. Further studies revealed that SI treatment did not alter GPX4 (glutathione peroxidase 4) expression, but led to the depletion of reduced thiol groups, mainly intracellular GSH (reduced glutathione) and cysteine. The study on iron trafficking demonstrated that iron influx was not altered by SI treatment but iron efflux increased, indicating that the increase in labile iron was likely due to the release of sequestered iron. This hypothesis was verified by showing that SI directly promoted the release of labile iron from a cell-free lysate. We propose that SI depletes GSH, increases ROS, releases labile iron, and boosts lipid damage, which in turn results in ferroptosis in ARPE-19 cells.

Two parameters of biological membranes, curvature and lipid composition, direct the recruitment of many peripheral proteins to cellular organelles. Although these traits are often studied independently, it is their combination that generates the unique interfacial properties of cellular membranes. Here, we use a combination of in vivo , in vitro and in silico approaches to provide a comprehensive map of how these parameters modulate membrane adhesive properties. The correlation between the membrane partitioning of model amphipathic helices and the distribution of lipid-packing defects in membranes of different shape and composition explains how macroscopic membrane properties modulate protein recruitment by changing the molecular topography of the membrane interfacial region. Furthermore, our results suggest that the range of conditions that can be obtained in a cellular context is remarkably large because lipid composition and curvature have, under most circumstances, cumulative effects. Membrane curvature and lipid composition direct the binding of many peripheral membrane proteins. Here, Vanni et al. use a combination of in vitro and molecular dynamics approaches to quantify how lipid-packing defects in membranes of various composition and curvature dictate the membrane adsorption of a model lipid-binding protein.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Dry AMD has unclear etiology and no treatment. Lipid-rich drusen are the hallmark of dry AMD. An AMD mouse model and insights into drusenogenesis are keys to better understanding of this disease. Chloride intracellular channel 4 (CLIC4) is a pleomorphic protein regulating diverse biological functions. Here we show that retinal pigment epithelium (RPE)-specific Clic4 knockout mice exhibit a full spectrum of functional and pathological hallmarks of dry AMD. Multidisciplinary longitudinal studies of disease progression in these mice support a mechanistic model that links RPE cell-autonomous aberrant lipid metabolism and transport to drusen formation. Age-related macular degeneration (AMD) is a leading cause of blindness and is characterised by the accumulation of lipid deposits, called drusen. Here, the authors show that mice lacking chloride intracellular channel 4 in retinal pigment epithelium have defective lipid processing in the eye and pathological features mirroring human AMD, including drusen formation.

Spermatogenesis, which is a continuous process from undifferentiated spermatogonia to spermatozoa in the seminiferous tubules, declines with age. To investigate changes in spermatogenesis with aging, we reconstructed the seminiferous tubules of 12 mice aged 12 to 30 months from serial sections and examined age-related and region-specific alterations in the seminiferous epithelium and spermatogenic waves in three dimensions. The basic structure of the seminiferous tubules, including the numbers of tubules, terminating points, branching points, and total tubule length, did not change with age. Age-related alterations in spermatogenesis, primarily assessed by the formation of vacuoles in Sertoli cells, were detected in the seminiferous tubules at 12 months. The proportion of altered tubule segments with impaired spermatogenesis further increased by 24 months, but remained unchanged thereafter. Altered tubule segments were preferentially distributed in tubule areas close to the rete testis and those in the center of the testis. Spermatogenic waves became shorter in length with age. These results provide a basis for examining the decline of spermatogenesis not only with aging, but also in male infertility.