Biomedical subjects

Junko Kitamoto

Publications and source records attributed to Junko Kitamoto.

8 recordsLinked to original sources

PubMed2005

Temporal regulation of global gene expression and cellular morphology in Xenopus kidney cells in response to clinorotation.

Here, we report changes gene expression and morphology of the renal epithelial cell line, A6, which was derived from Xenopus laevis adult kidney that had been induced by long-term culturing with a three-dimensional clinostat. An oligo microarray analysis on the A6 cells showed that mRNA levels for 52 out of 8091 genes were significantly altered in response to clinorotation. On day 5, there was no dramatic change in expression level, but by day 8 and day 10, either upregulation or downregulation of gene expression became evident. By day 15, the expression levels of 18 out of 52 genes had returned to the original levels, while the remaining 34 genes maintained the altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in the mRNA levels of selected genes were found only under clinorotation and not under hypergravity (7 g) or ground control. Morphological changes including loss of dome-like structures and disorganization of both E-cadherin adherence junctions and cortical actin were also observed after 10 days of culturing with clinorotation. These results revealed that the expression of selected genes was altered specifically in A6 cells cultured under clinorotation.

Animals↗

PubMed2005

Myosin VI is required for normal retinal function.

Different unconventional myosins have been shown to play important roles in sensory function, including vision. We investigated the role of myosin VI by examining the retinas of mice carrying a null mutation in the myosin VI gene. Myosin VI was found to be present in the photoreceptor and RPE cells of normal retinas. In the absence of myosin VI, the amplitudes of the a- and b-waves of the electroretinogram were reduced, although there was not photoreceptor cell loss and retinal anatomy appeared normal. Our results indicate that myosin VI is required in photoreceptor cells for normal retinal electrophysiology.

Animals↗

PubMed2004

Role of myosin VIIa and Rab27a in the motility and localization of RPE melanosomes.

Myosin VIIa functions in the outer retina, and loss of this function causes human blindness in Usher syndrome type 1B (USH1B). In mice with mutant Myo7a, melanosomes in the retinal pigmented epithelium (RPE) are distributed abnormally. In this investigation we detected many proteins in RPE cells that could potentially participate in melanosome transport, but of those tested, only myosin VIIa and Rab27a were found to be required for normal distribution. Two other expressed proteins, melanophilin and myosin Va, both of which are required for normal melanosome distribution in melanocytes, were not required in RPE, despite the association of myosin Va with the RPE melanosome fraction. Both myosin VIIa and myosin Va were immunodetected broadly in sections of the RPE, overlapping with a region of apical filamentous actin. Some 70-80% of the myosin VIIa in RPE cells was detected on melanosome membranes by both subcellular fractionation of RPE cells and quantitative immunoelectron microscopy, consistent with a role for myosin VIIa in melanosome motility. Time-lapse microscopy of melanosomes in primary cultures of mouse RPE cells demonstrated that the melanosomes move in a saltatory manner, interrupting slow movements with short bursts of rapid movement (>1 RR01183m/second). In RPE cells from Myo7a-null mice, both the slow and rapid movements still occurred, except that more melanosomes underwent rapid movements, and each movement extended approximately five times longer (and further). Hence, our studies demonstrate the presence of many potential effectors of melanosome motility and localization in the RPE, with a specific requirement for Rab27a and myosin VIIa, which function by transporting and constraining melanosomes within a region of filamentous actin. The presence of two distinct melanosome velocities in both control and Myo7a-null RPE cells suggests the involvement of at least two motors other than myosin VIIa in melanosome motility, most probably, a microtubule motor and myosin Va.

Animals↗

PubMed2004

Myosin Va is required for normal photoreceptor synaptic activity.

Myosin Va is an actin-based motor molecule, one of a large family of unconventional myosins. In humans, mutations in MYO5A cause Griscelli syndrome type 1 and Elejalde syndrome, diseases characterized by pigmentation defects and the prepubescent onset of severe neurological deficits that ultimately lead to a shortened lifespan. Mutations in the Myo5a gene in mouse cause the dilute series of mouse mutants, demonstrating that myosin Va is involved in pigmentation and neural function. Although the reason for the pigmentation abnormalities is well understood, the role of myosin Va in neural function is not. Myosin Va has been found in synaptic terminals in the retina and brain. We report here new physiological evidence for a role of myosin Va in synaptic function. Photoreceptor synapses in neurologically affected myosin Va mutant mice have both anatomical and physiological abnormalities. Thus, myosin Va is required for normal photoreceptor signalling, suggesting that it might function in central nervous system synapses in general, with aberrant synaptic activity potentially underlying the neurological defects observed in dilute lethal mice and patients with Griscelli syndrome type 1 and Elejalde syndrome.

Animals↗

PubMed2004

Global and temporal regulation of gene expression in Xenopus kidney cells in response to presumed microgravity generated by 3D clinostats.

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.

Actins↗

PubMed2003

Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.

Mutations in the myosin VIIa gene (MYO7A) cause Usher syndrome type 1B (USH1B), a major type of the deaf-blind disorder, Usher syndrome. We have studied mutant phenotypes in the retinas of Myo7a mutant mice (shaker1), with the aim of elucidating the role(s) of myosin VIIa in the retina and what might underlie photoreceptor degeneration in USH1B patients. A photoreceptor defect has been described. Here, we report that the phagocytosis of photoreceptor outer segment disks by the retinal pigment epithelium (RPE) is abnormal in Myo7a null mice. Both in vivo and in primary cultures of RPE cells, the transport of ingested disks out of the apical region is inhibited in the absence of Myo7a. The results with the cultured RPE cells were the same, irrespective of whether the disks came from wild-type or mutant mice, thus demonstrating that the RPE is the source of this defect. The inhibited transport seems to delay phagosome-lysosomal fusion, as the degradation of ingested disks was slower in mutant RPE. Moreover, fewer packets of disk membranes were ingested in vivo, possibly because retarded removal of phagosomes from the apical processes inhibited the ingestion of additional disk membranes. We conclude that Myo7a is required for the normal processing of ingested disk membranes in the RPE, primarily in the basal transport of phagosomes into the cell body where they then fuse with lysosomes. Because the phagocytosis of photoreceptor disks by the RPE has been shown to be critical for photoreceptor cell viability, this defect likely contributes to the progressive blindness in USH1B.

Animals↗

PubMed2003

Mouse models for Usher syndrome 1B.

Photoreceptor cell degeneration was not detected in any of the shaker1 alleles, except for a small but significant loss of photoreceptor cells found in Myo7a(4626SB/4626SB) mice that were also homozygous mutant for Cdh23v. Perhaps greater and/or faster photoreceptor cell loss that is dependent on mutant Myo7a can be effected by having additional mutant USH1 genes in the genetic background. In any case, it is argued that shaker1 mice are a useful model for testing USH1B gene therapy, due to the presence of mutant phenotypes other than photoreceptor cell death.

Alleles↗

PubMed2003

Cdh23 mutations in the mouse are associated with retinal dysfunction but not retinal degeneration.

Mutations in the cadherin 23 gene (CDH23) cause Usher syndrome type 1D in humans, a disease that results in retinitis pigmentosa and deafness. Cdh23 is also mutated in the waltzer mouse. In order to determine if the retina of the waltzer mouse undergoes retinal degeneration and to gain insight into the function of cadherin 23 in the retina, we have characterized the anatomy and physiology of retinas of waltzer mouse mutants. Three mutant alleles of Cdh23 were examined by histology and electroretinography (ERG). ERGs of the three Cdh23 mutant groups revealed two of them to have abnormal retinal function. One allele had a- and b-waves that were only approximately 80% of Cdh23 heterozygotes. Another allele had a significantly faster implicit time for both the a- and b-waves of the ERG. No anatomical abnormality was detected in any of the Cdh23 mutants by light microscopy. Because the mutant Cdh23 phenotype was found to be similar to the previously reported retinal phenotype of Myo7a mutant mice, the orthologue of another Usher syndrome (type 1B) gene, we generated mice that carried mutations in both genes to test for genetic interaction in the retina. No functional interaction between cadherin 23 and myosin VIIa was detected by either microscopy or ERG.

Alleles↗