Biology subjects

Spencer, W. J.

Publications and source records attributed to Spencer, W. J..

5 recordsLinked to original sources

bioRxiv2026

Rho/ROCK-dependent actomyosin contractility drives extracellular vesicle release from the cilium

The release of extracellular vesicles (EV) from the primary cilium is a conserved process observed in many cell types. It serves as a rapid and efficient mechanism to release select proteins from the cilium, which can be used for either intercellular communication or membrane material disposal. Previous studies have shown that the release of EVs from the cilium relies on the actin cytoskeleton and proposed several molecular mechanisms that may perform this function. Using the model of IMCD3 cells, we now demonstrate that this process relies on actomyosin contractility supported by non-muscle myosin IIA acting downstream of the RhoA-ROCK signaling pathway. We further showed that the cilia of these cells release EVs independently of de novo actin polymerization, which we confirmed using an in vivo model of mutant photoreceptor cells that release massive amounts of vesicles from their cilia instead of elaborating into light-sensitive outer segment membrane structures.

cell biology↗

bioRxiv2025

The tetraspanin disc proteins, peripherin-2 and ROM1, facilitate CNG channel localization to the rod outer segment

The light-responsive outer segment of rod photoreceptors is composed of two distinct membrane subdomains: discs and the plasma membrane. We investigate how the disc protein peripherin-2 is engaged in CNG channel delivery to the outer segment. Instead of forming outer segments, peripherin-2 knockout (Rds-/-) photoreceptors release ciliary ectosomes, in which CNG channel levels are markedly reduced relative to other outer segment proteins. This is intriguing as downregulation of the CNG channel is not a general feature of degenerative mouse models with dysmorphic outer segments. Overexpression of the {beta}1-subunit of CNG in Rds-/- rods reveals that the majority is trapped in intracellular membranes, but is restored to the outer segment by co-expressing peripherin-2. We test peripherin-2 chimeras containing either the N-terminus, tetraspanin core, or C-terminus and find that the tetraspanin domain is sufficient to localize CNG{beta}1 to the outer segment. We further show that the membrane remodeling function of the tetraspanin domain facilitates this process by the redundant action of the tetraspanin domain from ROM1 and the reemergence of the endogenous CNG channel in aged Rds-/- rods that have produced ciliary membrane protrusions.

cell biology↗

bioRxiv2024

Inpp5e is a Critical Regulator of Protein Transport to Photoreceptor Outer Segments

In humans, inositol polyphosphate-5-phosphatase e (INPP5E) mutations cause retinal degeneration as part of Joubert and MORM syndromes and can also cause non-syndromic blindness. In mice, mutations cause a spectrum of brain, kidney, and other anomalies and prevent the formation of photoreceptor outer segments. To further explore the function of Inpp5e in photoreceptors, we generated conditional and inducible knockouts of mouse Inpp5e where the gene was deleted either during outer segment formation or after outer segments were fully formed. In both cases, the loss of Inpp5e led to severe defects in photoreceptor outer segment morphology and ultimately photoreceptor cell loss. The primary morphological defect consisted of outer segment shortening and reduction in the number of newly forming discs at the outer segment base. This was accompanied by structural abnormalities of the Golgi apparatus, mislocalized rhodopsin, and an accumulation of extracellular vesicles. In addition, knockout cells showed a reduction in the size and prevalence of the actin network at the site of new disc morphogenesis and the occasional formation of membrane whorls instead of discs in a subset of cells. Together, these data demonstrate that Inpp5e plays a critical role in maintaining the outer segment and the normal process of outer segment renewal depends on the activity of this enzyme.

cell biology↗

bioRxiv2023

Absolute quantification of photoreceptor outer segment proteins

Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place in a highly specialized outer segment organelle. There are significant discrepancies in the reported amounts of many proteins supporting this process, particularly those of low abundance, which limits our understanding of their molecular organization and function. In this study, we used quantitative mass spectrometry to simultaneously determine the abundances of twenty key structural and functional proteins residing in mouse rod outer segments. We computed the absolute number of molecules of each protein residing within an individual outer segment and the molar ratio amongst all twenty proteins. The molar ratios of proteins comprising three well-characterized constitutive complexes in outer segments differed from the established subunit stoichiometries of these complexes by less than 7%, highlighting the exceptional precision of our quantification. Overall, this study resolves multiple existing discrepancies regarding the outer segment abundances of these proteins, thereby advancing our understanding of how the phototransduction pathway functions as a single, well-coordinated molecular ensemble.

biochemistry↗

bioRxiv2022

The WAVE complex drives the morphogenesis of the photoreceptor outer segment cilium

The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain photoreceptor health and functionality, outer segments are continuously renewed through the addition of new discs at their base. This process is driven by branched actin polymerization nucleated by the Arp2/3 complex. To induce actin polymerization, Arp2/3 requires a nucleation promoting factor. Here, we show that the nucleation promoting factor driving disc morphogenesis is the pentameric WAVE complex and identify all protein subunits of this complex. We further demonstrate that the knockout of one of them, WASF3, abolishes actin polymerization at the site of disc morphogenesis leading to formation of disorganized membrane lamellae emanating from the photoreceptor cilium instead of an outer segment. These data establish that, despite the intrinsic ability of photoreceptor ciliary membranes to form lamellar structures, WAVE-dependent actin polymerization is essential for organizing these membranes into a proper outer segment.

cell biology↗