MAST4 promotes primary ciliary resorption through phosphorylation of Tctex-1.

The primary cilium undergoes cell cycle-dependent assembly and disassembly. Dysregulated ciliary dynamics are associated with several pathological conditions called ciliopathies. Previous studies showed that the localization of phosphorylated Tctex-1 at Thr94 (T94) at the ciliary base critically regulates ciliary resorption by accelerating actin remodeling and ciliary pocket membrane endocytosis.

Non-reversible tissue fixation retains extracellular vesicles for in situ imaging.

Extracellular vesicles (EVs) are secreted nanosized particles with many biological functions and pathological associations. The inability to image EVs in fixed tissues has been a major limitation to understanding their role in healthy and diseased tissue microenvironments. Here, we show that crosslinking mammalian tissues with formaldehyde results in significant EV loss, which can be prevented by additional fixation with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for visualization of EVs in a range of normal and cancer tissues.

[Mechanisms of cell proliferation through primary cilium].

Primary cilium is a membrane-protruding sensory organelle, which is organized from a basal body in G/G phase cells. The resorption of primary cilia under specific growth factor stimuli is coupled to cell cycle re-entry and cell proliferation, and the proliferative function is vital for the organization of organs at an embryonic stage. In fact, abnormalities in ciliogenesis and/or cilium-derived signaling lead to malformation of various organs, such as the brain, eyes, nose, ear, heart, lung, liver, kidney, and bones.

Ciliary Assembly/Disassembly Assay in Non-transformed Cell Lines.

The primary cilium is a non-motile sensory organelle whose assembly and disassembly are closely associated with cell cycle progression. The primary cilium is elongated from the basal body in quiescent cells and is resorbed as the cells re-enter the cell cycle. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases.

Tctex-1 controls ciliary resorption by regulating branched actin polymerization and endocytosis.

The primary cilium is a plasma membrane-protruding sensory organelle that undergoes regulated assembly and resorption. While the assembly process has been studied extensively, the cellular machinery that governs ciliary resorption is less well understood. Previous studies showed that the ciliary pocket membrane is an actin-rich, endocytosis-active periciliary subdomain.

The Biology of Ciliary Dynamics.

The cilium is an evolutionally conserved apical membrane protrusion that senses and transduces diverse signals to regulate a wide range of cellular activities. The cilium is dynamic in length, structure, and protein composition. Dysregulation of ciliary dynamics has been linked with ciliopathies and other human diseases.

Degradation of silk films in multipocket corneal stromal rabbit models.

Introduction: The need for human cornea tissues continues to grow as an alternative option to donor tissues. Silk protein has been successfully used as a substrate to engineer corneal epithelium and stroma in vitro. Herein, we investigated the in vivo response and the effect of silk crystalline structure (beta sheet) on degradation rate of silk films in rabbit multipocket corneal models.

The Disease Protein Tulp1 Is Essential for Periactive Zone Endocytosis in Photoreceptor Ribbon Synapses.

Mutations in the Tulp1 gene cause severe, early-onset retinitis pigmentosa (RP14) in humans. In the retina, Tulp1 is mainly expressed in photoreceptors that use ribbon synapses to communicate with the inner retina. In the present study, we demonstrate that Tulp1 is highly enriched in the periactive zone of photoreceptor presynaptic terminals where Tulp1 colocalizes with major endocytic proteins close to the synaptic ribbon.

Ultrastructural visualization of trans-ciliary rhodopsin cargoes in mammalian rods.

Background: Cilia are vital to various cellular and sensory functions. The pathway by which ciliary membrane proteins translocate through the transition zone is not well understood. Direct morphological characterization of ciliary cargoes in transit remains lacking.

VEGF-B selectively regenerates injured peripheral neurons and restores sensory and trophic functions.

VEGF-B primarily provides neuroprotection and improves survival in CNS-derived neurons. However, its actions on the peripheral nervous system have been less characterized. We examined whether VEGF-B mediates peripheral nerve repair.

Characterization of oxygen-induced retinopathy in mice carrying an inactivating point mutation in the catalytic site of ADAM15.

Purpose: Retinal neovascularization is found in diseases such as macular degeneration, diabetic retinopathy, or retinopathy of prematurity and is usually caused by alterations in oxygen supply. We have previously described that mice lacking the membrane-anchored metalloproteinase ADAM15 (a Disintegrin and Metalloprotease 15) have decreased pathological neovascularization of the retina in the oxygen-induced retinopathy (OIR) model. The main purpose of the present study was to determine the contribution of the catalytic activity of ADAM15 to OIR.

Synthetic neurotensin analogues are nontoxic analgesics for the rabbit cornea.

Purpose: To characterize the analgesic potency and toxicity of topical synthetic neurotensin analogues, and localize neurotensin receptors in the cornea and trigeminal ganglion.

Methods: Cochet-Bonnet esthesiometry was performed on the rabbit cornea to test the analgesic dose response and duration of effect for two synthetic neurotensin analogues: NT71 and NT72. Receptors for neurotensin were localized in the murine cornea and trigeminal ganglion using quantitative PCR (qPCR), Western blotting, and immunohistochemistry.

Four-dimensional live imaging of apical biosynthetic trafficking reveals a post-Golgi sorting role of apical endosomal intermediates.

Emerging data suggest that in polarized epithelial cells newly synthesized apical and basolateral plasma membrane proteins traffic through different endosomal compartments en route to the respective cell surface. However, direct evidence for trans-endosomal pathways of plasma membrane proteins is still missing and the mechanisms involved are poorly understood. Here, we imaged the entire biosynthetic route of rhodopsin-GFP, an apical marker in epithelial cells, synchronized through recombinant conditional aggregation domains, in live Madin-Darby canine kidney cells using spinning disk confocal microscopy.

The roles of evolutionarily conserved functional modules in cilia-related trafficking.

Cilia are present across most eukaryotic phyla and have diverse sensory and motility roles in animal physiology, cell signalling and development. Their biogenesis and maintenance depend on vesicular and intraciliary (intraflagellar) trafficking pathways that share conserved structural and functional modules. The functional units of the interconnected pathways, which include proteins involved in membrane coating as well as small GTPases and their accessory factors, were first experimentally associated with canonical vesicular trafficking.

IGF-1 activates a cilium-localized noncanonical Gβγ signaling pathway that regulates cell-cycle progression.

Primary cilia undergo cell-cycle-dependent assembly and disassembly. Emerging data suggest that ciliary resorption is a checkpoint for S phase reentry and that the activation of phospho(T94)Tctex-1 couples these two events. However, the environmental cues and molecular mechanisms that trigger these processes remain unknown.

Vascular endothelial growth factor promotes anatomical and functional recovery of injured peripheral nerves in the avascular cornea.

Peripheral nerve injury is a major neurological disorder that can cause severe motor and sensory dysfunction. Neurogenic effects of vascular endothelial growth factor (VEGF) have been found in the central nervous system, and we examined whether VEGF could promote anatomical and functional recovery of peripheral nerves after injury using an avascular corneal nerve injury model. We found that VEGF enhanced neurite elongation in isolated trigeminal ganglion neurons in a dose-dependent manner.

Silk film culture system for in vitro analysis and biomaterial design.

Silk films are promising protein-based biomaterials that can be fabricated with high fidelity and economically within a research laboratory environment (1,2). These materials are desirable because they possess highly controllable dimensional and material characteristics, are biocompatible and promote cell adhesion, can be modified through topographic patterning or by chemically altering the surface, and can be used as a depot for biologically active molecules for drug delivery related applications (3-8). In addition, silk films are relatively straightforward to custom design, can be designed to dissolve within minutes or degrade over years in vitro or in vivo, and are produce with the added benefit of being transparent in nature and therefore highly suitable for imaging applications (9-13).

Retinal pigment epithelial cells use a MerTK-dependent mechanism to limit the phagocytic particle binding activity of αvβ5 integrin.

Background Information: αvβ5 integrin and Mer tyrosine kinase (MerTK) receptors reside at the apical surface of the retinal pigment epithelium (RPE) in the eye to promote the diurnal, synchronised phagocytosis of shed photoreceptor outer segment fragments (POS) that is critical for vision. Phagocytosis assays studying RPE cells in culture have defined roles for αvβ5 in POS surface binding and for MerTK in engulfment of bound POS. Both receptors have thus far only been studied separately.

Ciliary transition zone activation of phosphorylated Tctex-1 controls ciliary resorption, S-phase entry and fate of neural progenitors.

Primary cilia are displayed during the G(0)/G(1) phase of many cell types. Cilia are resorbed as cells prepare to re-enter the cell cycle, but the causal and molecular link between these two cellular events remains unclear. We show that Tctex-1 phosphorylated at Thr 94 is recruited to ciliary transition zones before S-phase entry and has a pivotal role in both ciliary disassembly and cell cycle progression.

Identification of the Tctex-1 regulatory element that directs expression to neural stem/progenitor cells in developing and adult brain.

Previous studies showed that Tctex-1 immunoreactivity is selectively enriched in the germinal zones of adult brain. In this report we identify a regulatory region of the Tctex-1 gene that is capable of directing transgenic expression of green fluorescent protein (GFP) reporter that recapitulates the spatial and temporal expression pattern of endogenous Tctex-1. This construct specifically targeted expression to the nestin(+)/Pax6(+)/GLAST(+) radial glial cells and Tbr2(+) intermediate progenitors when the reporter construct was delivered to developing mouse neocortex via in utero electroporation.

It takes two to tango to the melanosome.

The role of clathrin adaptor proteins in sorting cargo in the biosynthetic and recycling routes is an area of intense research. In this issue, Delevoye et al. (2009.

Itinerant exosomes: emerging roles in cell and tissue polarity.

Cells use secreted signals (e.g. chemokines and growth factors) and sophisticated vehicles such as argosomes, cytonemes, tunneling nanotubes and exosomes to relay important information to other cells, often over large distances.