Vertebrate and invertebrate photoreceptors shed their photosensitive membrane on a daily basis. Although we have detailed knowledge of the morphology of the disc shedding and renewal process in vertebrate photoreceptors, and of the turnover of rhabdom in invertebrate photoreceptors, we know relatively little about the molecular mechanisms whereby these processes are triggered by light and/or by circadian efferent input to the retina. We have used the horseshoe crab, Limulus polyphemus, as a model system to unravel the molecular means by which the trigger light is communicated to the intracellular machinery responsible for the daily breakdown of the photosensitive membrane. Phorbol esters, potent and specific activators of protein kinase C (PKC), induce a robust burst of rhabdom shedding when injected subretinally into the compound lateral eye of Limulus. This occurs in the absence of the light trigger normally required to initiate shedding in the lateral eye at dawn, suggesting that PKC may play a role in the light triggering of rhabdom shedding. Diacylglycerol (DAG) analogs were also found to elicit rhabdom shedding in the lateral eye without a light trigger, but at uncharacteristically high concentrations. However, injecting inositol trisphosphate (InsP3) and DAG analog simultaneously results in a tenfold decrease in the concentration of DAG analog required to initiate a shedding event. Immunohistochemical screening for PKC in the lateral eye shows that two isozymes (PKC beta II and PKC zeta) are co-localized to the retinular cell rhabdom. Taken together, these data suggest that light triggers rhabdom shedding at dawn via a classical Ca(2+)-sensitive PKC, similar to PKC beta II, which is activated synergistically by the light-evoked production of DAG and InsP3.
Administration of chlorpromazine inhibits the antidiuretic response of rats to painful stimuli but has no significant effect on antidiuretic response to injection of either histamine or nicotine.
Abstract Enzymatic treatments that facilitated whole-cell electrophysiological recordings were used on Limulus ventral photoreceptor cells. Ventral optic nerves were treated with either collagenase or collagenase, papain, and trypsin. Either treatment greatly increased the ease of making whole-cell recordings of transmembrane potentials. Light responses obtained from enzyme-treated photoreceptor cells were nearly identical to results obtained without enzyme treatment and compared favorably to in vivo recordings of light responses from the compound lateral eye. Enzyme-treated cells also responded to applied octopamine, as do untreated cells, with an increased phosphorylation of a 122-kD protein. This suggests that the external receptors and internal biochemical machinery required for at least one second-messenger cascade are present after enzyme treatment. The morphological integrity of enzyme-treated photoreceptor cells was examined with light microscopy as well as with scanning and transmission electron microscopy. In general, we found that each enzyme treatment greatly reduced the integrity of the layers of glial cells that surround the photoreceptor cells thereby making these cells easily accessible for whole-cell recordings of transmembrane potentials. The morphology of the rhabdomere was normal after enzymatic degradation of the adjacent glial covering.
Actin nucleation factors function to organize, shape, and move membrane-bound organelles, yet they remain poorly defined in relation to disease. Galloway-Mowat syndrome (GMS) is an inherited disorder characterized by microcephaly and nephrosis resulting from mutations in the WDR73 gene. This core clinical phenotype appears frequently in the Amish, where virtually all affected individuals harbor homozygous founder mutations in WDR73 as well as the closely linked WHAMM gene, which encodes a nucleation factor. Here we show that patient cells with both mutations exhibit cytoskeletal irregularities and severe defects in autophagy. Reintroduction of wild-type WHAMM restored autophagosomal biogenesis to patient cells, while inactivation of WHAMM in healthy cell lines inhibited lipidation of the autophagosomal protein LC3 and clearance of ubiquitinated protein aggregates. Normal WHAMM function involved binding to the phospholipid PI(3)P and promoting actin nucleation at nascent autophagosomes. These results reveal a cytoskeletal pathway controlling autophagosomal remodeling and illustrate several molecular processes that are perturbed in Amish GMS patients.
Synthetic oxytocin and analogues of oxytocin exert an insulinlike action on the utilization of glucose by rat epididymal adipose tissue. The insulinlike action appears to be dependent upon the factors that determine the oxytocic properties of the peptides.
Next‐generation sequencing accelerates disease gene discovery, especially for orphan diseases, though at present it outpaces functional studies needed to provide ‘proof of causation.’ Working from a small, community‐based, clinical laboratory, the Clinic for Special Children has identified more than 170 allelic variants associated with disability, disease, or untimely death among the Amish and Mennonite (Plain) populations of North America. In collaboration with the Clinic, we developed an HHMI‐funded program that integrates functional studies of novel disease gene alleles into our undergraduate curriculum. Roughly 150 students per year in our introductory cell biology and neuroscience courses clone human disease genes and study the functional impacts of gene variants through expression in mammalian cell culture. We use these authentic research experiences to teach key concepts in cell biology, genetics, and neuroscience. Students later build upon this experience in upper‐level courses in cell biology, neuroscience, genetics, cancer biology, and immunology in which they engage in semester‐long research projects in small teams. Teams conduct functional studies of disease gene variants or study novel cellular or animal models of disease. These experiences engender talented undergraduates to assume greater research responsibility through independent study and summer research projects that transition them into the role of co‐PI for their project. Data published through this project ( e.g., PLoS ONE 7:e28936) have been used by other institutions to establish novel diagnoses, including a high‐profile diagnosis of a critically‐ill, non‐Plain newborn with lethal neonatal seizure‐rigidity syndrome ( Sci Transl Med 4:154ra135). This provides important proof‐of‐concept for integrating novel disease gene functional studies into a carefully structured undergraduate research curriculum. Students in our Public Health program are collaborating with the Clinic to develop handbooks to help parents care for children with special medical needs with the goal of producing one to two high‐quality disease handbooks annually. Our program represents a model for engaging undergraduates in meaningful research at the front lines of biomedical science and public health in ways that directly impact the diagnosis and care of children with rare inherited disorders and promote STEM retention. Support or Funding Information HHMI Undergraduate Science Education Awards 52006294 and 52007538.
THE exposure of normal rats to a variety of noxious stimuli (pain, noise, a strange environment, and the intraperitoneal injection of histamine) results in a rapid, marked increase in the antidiuretic activity of the plasma (Mirsky, Stein and Paulisch, 1953). Although the precise nature of the substance (or substances) (ADS) responsible for the augmented antidiuretic activity was not established, its resemblance to the antidiuretic hormone (ADH) extractable from the neurohypophysis and the speed with which it is released into the circulation suggested that ADS and ADH may be identical. It was proposed that common to the specific reactions induced by the various noxious stimuli is an activation of the hypothalamus with a resultant release of the antidiuretic hormone into the circulation. It is generally assumed that the secretion of the antidiuretic hormone is dependent upon the integrity of the supraoptic and para ventricular nuclei, the supraoptico-hypophyseal tract and the neurohypophysis
Abstract Examination of the Limulus median optic nerve with low-magnification light microscopy allows clear visualization of an ultraviolet-sensitive mini-ommatidium enshrouded by pigment cells, glial cells, and guanophores. Serial 1-μm sections of median optic nerves containing mini-ommatidia revealed the presence of a single, heavily pigmented photoreceptor (retinular) cell and a single, unpigmented arhabdomeric cell. Computer-assisted serial reconstructions from 1-μm sections confirmed the presence of two cells, each bearing a nucleus, and two axons leaving the mini-ommatidium. The retinular cell is morphologically similar to retinular cells from the median and lateral eyes. Its rhabdomere appears to be a continuous sheet of microvilli with much infolding. The structure of the arhabdomeric cell is nearly identical to those found in the median ocellus. As in other photoreceptors in Limulus , the retinular cell of the mini-ommatidium is innervated by efferent fibers from the brain. Each mini-ommatidium generates a single train of nerve impulses in response to light, presumably from the arhabdomeric cell. Measurement of the spectral sensitivity of the mini-ommatidium based upon a constant-response criterion indicated that the retinular cell is maximally sensitive to near ultraviolet light with λ max = 380 nm. Comparison of intensity-response functions revealed that those of the mini-ommatidium are significantly steeper than those of the ocellus almost certainly as the result of neural processing in the ocellus which is absent in the mini-ommatidium.
