Loss of Motor Protein MYO1C Causes Rhodopsin Mislocalization and Results in Impaired Visual Function

Unconventional myosins, linked to deafness, are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice, specifically causes rhodopsin mislocalization, leading to impaired visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with MYO1C. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is important for rhodopsin localization to the photoreceptor OS, and for normal visual function.

A Systemic and Molecular Study of Subcellular Localization of SARS-CoV-2 Proteins

Coronavirus possesses the largest RNA genome among all the RNA viruses. Its genome encodes about 29 proteins. Most of the viral proteins are non-structural proteins (NSP) except envelop (E), membrane (M), nucleocapsid (N) and Spike (S) proteins that constitute the viral nucleocapsid, envelop and surface. We have recently cloned all the 29 SARS-CoV-2 genes into vectors for their expressions in mammalian cells except NSP11 that has only 14 amino acids (aa). We are able to express all the 28 cloned SARS-CoV-2 genes in human cells to characterize their subcellular distributions. The proteins of SARS-CoV-2 are mostly cytoplasmic but some are both cytoplasmic and nuclear. Those punctate staining proteins were further investigated by immunofluorescent assay (IFA) using specific antibodies or by co-transfection with an organelle marker-expressing plasmid. As a result, we found that NSP15, ORF6, M and ORF7a are related to Golgi apparatus, and that ORF7b, ORF8 and ORF10 colocalize with endoplasmic reticulum (ER). Interestingly, ORF3a distributes in cell membrane, early endosome, endosome, late endosome and lysosome, which suggests that ORF3a might help the infected virus to usurp endosome and lysosome for viral use. Furthermore, we revealed that NSP13 colocalized with SC35, a protein standing for splicing compartments in the nucleus. Our studies for the first time visualized the subcellular locations of SARS-CoV-2 proteins and might provide novel insights into the viral proteins’ biological functions.

Motor Protein MYO1C is Critical for Photoreceptor Opsin Trafficking and Visual Function

Unconventional myosins linked to deafness are also proposed to play a role in retinal cell physiology. However, their direct role in photoreceptor function remains unclear. We demonstrate that systemic loss of the unconventional myosin MYO1C in mice specifically affected opsin trafficking, leading to loss of visual function. Electroretinogram analysis of Myo1c knockout (Myo1c-KO) mice showed a progressive loss of photoreceptor function. Immunohistochemistry and binding assays demonstrated MYO1C localization to photoreceptor inner and outer segments (OS) and identified a direct interaction of rhodopsin with the MYO1C cargo domain. In Myo1c-KO retinas, rhodopsin mislocalized to rod inner segments (IS) and cell bodies, while cone opsins in OS showed punctate staining. In aged mice, the histological and ultrastructural examination of the phenotype of Myo1c-KO retinas showed progressively shorter photoreceptor OS. These results demonstrate that MYO1C is critical for opsin trafficking to the photoreceptor OS and for normal visual function.

Pure Discrete Punctate Nuclear Staining Pattern for MLH1 Protein Does Not Represent Intact Nuclear Expression

Immunohistochemical staining for DNA mismatch repair (MMR) proteins is commonly used to screen for Lynch syndrome. Several laboratories have noticed a discrete punctate nuclear staining pattern for MLH1 that caused confusion in interpretation. This study was designed to investigate whether this particular staining pattern represents intact nuclear expression of MLH1. MMR proteins immunostaining and follow-up testing in 161 consecutive colorectal adenocarcinoma cases (86 biopsies, 75 resections) were retrospectively reviewed. Both discrete punctate nuclear staining and diffuse nuclear staining patterns for MLH1 were observed in internal control cells in 76 biopsies and 27 resections. Only diffuse nuclear staining was seen in the remaining 10 biopsies and 48 resections ( P < .0001). Pure discrete punctate nuclear staining pattern for MLH1 was observed in 11 tumors (9 biopsies, 2 resections), and completely negative staining was seen in 13 tumors (2 biopsies, 11 resections; P = .003). Those 24 tumors (21 patients) invariably showed loss of PMS2. Three patients whose biopsies showed pure punctate staining for MLH1 underwent repeat testing on resections: 1 retained the punctate staining and 2 showed complete loss of MLH1. Nine patients who showed loss of PMS2 and pure punctate MLH1 staining underwent molecular testing: 4 had BRAF V600E mutations and 1 had MLH1 gene mutation. Our data showed that discrete punctate nuclear staining for MLH1 is more commonly seen in biopsy specimens. Pure discrete punctate staining pattern is paired with loss of PMS2 expression and may be associated with BRAF or MLH1 gene mutation, thus it should not be interpreted as intact nuclear expression.

Identification of a Cytoplasmic Complex That Adds a Cap onto 5′-Monophosphate RNA

ABSTRACT Endonuclease decay of nonsense-containing β-globin mRNA in erythroid cells generates 5′-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5′ modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5′-monophosphate RNA into 5′-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.

229CHARACTERIZATION OF CONNEXIN 43 IN THE CANINE OVARIAN FOLLICLE

Connexin 43 (Cx43) is the most abundant gap-junction protein in ovarian follicle cell interactions and has been well characterized for several mammalian species. However, the involvement of Cx43 in canine follicular development has not been extensively investigated. The objective of this study was to 1) characterize the spatial patterns of Cx43 localization within the canine ovarian follicle at various physiological states and during the estrous cycle (prepubertal, anestrus, proestrus, estrus and diestrus), and 2) to characterize the canine Cx43 mRNA transcript. The spatial expression pattern of Cx43 protein was evaluated by immunofluorescence microscopy in canine ovaries. Cx43 was not detected in primordial follicles, but was detected in primary and secondary follicles at each physiological state at all granulosa cell borders. In secondary follicles, definitive, punctate staining patterns were localized along mural granulosa cells and also in the surrounding granulosa-cumulus cell borders. Notably, more intense staining was observed in the corona radiata cells immediately surrounding the oocyte, as well as in trans-zonal projections and at the perivitelline membrane. Patterns of localization were most similar between proestrus and diestrus, and between prepubertal and anestrus in secondary follicles. Estrus-stage follicles showed a decrease in localization at the corona-oocyte cell borders as compared to proestrus and diestrus. In large, healthy antral follicles from proestrus and estrus stages, Cx43 was present in the stroma, theca, and granulosa layers. However, antral follicles from estrus-stage ovaries showed more intense staining in the mural granulosa and theca layers, and less intense in the stroma as compared to those of proestrus stage. The most intense pattern of punctate staining was observed in the corpora lutea of diestrus-stage ovaries. Additionally, gene-specific primers were designed from highly conserved regions of Cx43 mRNA among bovine, human and mouse. RNA was isolated from canine uterus and used as a template for RT-PCR. The PCR products were then sequenced and verified in GenBank to assess homology. The sequenced coding region for canine Cx43 mRNA shares 93% sequence homology with bovine vascular smooth muscle. Canine specific primers for this sequence have been designed, and expression analysis studies in canine ovarian follicles are currently underway. These results indicate that the pattern of localization of Cx43 is similar to that reported for the cow and the pig, except that in canine ovarian preantral follicles, Cx43 is also localized to the peri vitelline membrane. Additionally, these results suggest that the localization of Cx43 is dependent on the physiological state of the ovary, and is likely necessary for folliculogenesis and subsequent oocyte development in canines.

Subcellular distribution of ryanodine receptors in the cardiac muscle of carp (Cyprinus carpio)

We examined the subcellular localization of ryanodine receptors (RyR) in the cardiac muscle of carp using biochemical, immunohistochemical, and electron microscopic methods and compared it with those of rats and guinea pigs. To achieve this goal, an anti-RyR antibody was newly raised against a synthetic peptide corresponding to an amino acid sequence that was conserved among all sequenced RyRs. Western blot analysis using this antibody detected a single RyR band following the SDS-PAGE of sarcoplasmic reticulum (SR) membranes from carp atrium and ventricle as well as from mammalian hearts and skeletal muscles. The carp heart band had slightly greater mobility than those of mammalian hearts. Although immunohistochemical staining showed evident striations corresponding to the Z lines in longitudinal sections of mammalian hearts, clusters of punctate staining, in contrast, were distributed ubiquitously throughout carp atrium and ventricle. Electron microscopic images of the carp myocardium showed that the SR was observed largely as the subsarcolemmal cisternae and the reticular SR, suggesting that the RyR is localized in the junctional and corbular SR.