Hepatitis C virus (HCV) enters hepatocytes via various entry factors, including scavenger receptor BI (SR-B1), cluster of differentiation 81 (CD81), epidermal growth factor receptor (EGFR), claudin-1 (CLDN1), and occludin (OCLN). As CLDN1 and OCLN are not readily accessible due to their tight junctional localization, HCV likely accesses them by either disrupting cellular polarity or migrating to the tight junction. In this study, we image HCV entry into a three-dimensional polarized hepatoma system and reveal that the virus sequentially engages these entry factors through actin-dependent mechanisms. HCV initially localizes with the early entry factors SR-B1, CD81, and EGFR at the basolateral membrane and then accumulates at the tight junction in an actin-dependent manner. HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR. Thus, HCV uses a dynamic and multi-step process to engage and enter host cells.
Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss. The protein HtrA1 is enriched in retinal pigment epithelial (RPE) cells isolated from AMD patients and in drusen deposits. However, it is poorly understood how increased levels of HtrA1 affect the physiological function of the RPE at the intracellular level. Here, we developed hfRPE (human fetal retinal pigment epithelial) cell culture model where cells fully differentiated into a polarized functional monolayer. In this model, we fine-tuned the cellular levels of HtrA1 by targeted overexpression. Our data show that HtrA1 enzymatic activity leads to intracellular degradation of tubulin with a corresponding reduction in the number of microtubules, and consequently to an altered mechanical cell phenotype. HtrA1 overexpression further leads to impaired apical processes and decreased phagocytosis, an essential function for photoreceptor survival. These cellular alterations correlate with the AMD phenotype and thus highlight HtrA1 as an intracellular target for therapeutic interventions towards AMD treatment.
Two important events that occur during mammalian spermatogenesis are the release of elongated spermatids at late stage VIII of the seminiferous epithelial cycle and the restructuring of the blood-testis barrier (BTB) during stages VIII-XI. Still, it is not completely understood how these cellular events are accomplished within the seminiferous epithelium. In the present study, we investigate how sertolin, a protein that was initially identified, cloned, and partially characterized by our laboratory, functions in these critical events. Sertolin was found at the BTB, as well as at the apical ectoplasmic specialization and apical tubulobulbar complex, where it colocalized with epidermal growth factor receptor kinase substrate 8 and actin-related protein 3, two actin-regulatory proteins. Knockdown of sertolin by RNA interference showed Sertoli cell barrier function to be enhanced when assessed by transepithelial electrical resistance measurements and immunolocalization experiments. By contrast, the integrity of the BTB was disrupted when sertolin was overexpressed in vitro and in vivo. Sertolin overexpression also prompted germ cell loss from the seminiferous epithelium. Taken collectively, these results suggest that sertolin may be involved in coordinating spermatid release and BTB restructuring during spermatogenesis in the rat.