Deletion of myosin VI causes slow retinal optic neuropathy and age-related macular degeneration (AMD)-relevant retinal phenotype.
ABSTRACT: The unconventional myosin VI, a member of the actin-based motor protein family of myosins, is expressed in the retina. Its deletion was previously shown to reduce amplitudes of the a- and b-waves of the electroretinogram. Analyzing wild-type and myosin VI-deficient Snell's Waltzer mice in more detail, the expression pattern of myosin VI in retinal pigment epithelium, outer limiting membrane, and outer plexiform layer could be linked with differential progressing ocular deficits. These encompassed reduced a-waves and b-waves and disturbed oscillatory potentials in the electroretinogram, photoreceptor cell death, retinal microglia infiltration, and formation of basal laminar deposits. A phenotype comprising features of glaucoma (neurodegeneration) and age-related macular degeneration could thus be uncovered that suggests dysfunction of myosin VI and its variable cargo adaptor proteins for membrane sorting and autophagy, as possible candidate mediators for both disease forms.
Project description:Golgi morphology and function are dependent on an intact microtubule and actin cytoskeleton. Myosin VI, an unusual actin-based motor protein moving towards the minus ends of actin filaments, has been localized to the Golgi complex at the light and electron microscopic level. Myosin VI is present in purified Golgi membranes as a peripheral membrane protein, targeted by its globular tail domain. To investigate the function of myosin VI at the Golgi complex, immortal fibroblastic cell lines of Snell's waltzer mice lacking myosin VI were established. In these cell lines, where myosin VI is absent, the Golgi complex is reduced in size by approximately 40% compared with wild-type cells. Furthermore, protein secretion of a reporter protein from Snell's waltzer cells is also reduced by 40% compared with wild-type cells. Rescue experiments showed that fully functional myosin VI was able to restore Golgi complex morphology and protein secretion in Snell's waltzer cells to the same level as that observed in wild-type cells.
Project description:Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snell's waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.
Project description:The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and alpha-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether alpha-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with alpha-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to alpha-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with alpha-AP-2. siRNA silencing of alpha-AP-2 in cells significantly reduced CFTR endocytosis, further supporting alpha-AP-2 as the direct binding partner for CFTR. alpha-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell's Waltzer myosin VI((sv/sv)) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI((sv/sv)) enterocytes. The findings support alpha-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.
Project description:During spermiogenesis in mammals, actin filaments and a variety of actin-binding proteins are involved in the formation and function of highly specialized testis-specific structures. Actin-based motor proteins, such as myosin Va and VIIa, play a key role in this complex process of spermatid transformation into mature sperm. We have previously demonstrated that myosin VI (MYO6) is also expressed in mouse testes. It is present in actin-rich structures important for spermatid development, including one of the earliest events in spermiogenesis-acrosome formation. Here, we demonstrate using immunofluorescence, cytochemical, and ultrastructural approaches that MYO6 is involved in maintaining the structural integrity of these specialized actin-rich structures during acrosome biogenesis in mouse. We show that MYO6 together with its binding partner TOM1/L2 is present at/around the spermatid Golgi complex and the nascent acrosome. Depletion of MYO6 in Snell's waltzer mice causes structural disruptions of the Golgi complex and affects the acrosomal granule positioning within the developing acrosome. In summary, our results suggest that MYO6 plays an anchoring role during the acrosome biogenesis mainly by tethering of different cargo/membranes to highly specialized actin-related structures.
Project description:The action of extracellular protons on retinal activity and phototransduction occurs through pH-sensitive elements, mainly membrane conductances present on the different cell types of the outer and inner nuclear layers and of the ganglion cell layer. Acid-sensing ion channels (ASICs) are depolarizing conductances that are directly activated by protons. We investigated the participation of ASIC1a, a particular isoform of ASICs, in retinal physiology in vivo using electroretinogram measurements. In situ hybridization and immunohistochemistry localized ASIC1a in the outer and inner nuclear layers (cone photoreceptors, horizontal cells, some amacrine and bipolar cells) and in the ganglion cell layer. Both the in vivo knockdown of ASIC1a by antisense oligonucleotides and the in vivo blocking of its activity by PcTx1, a specific venom peptide, were able to decrease significantly and reversibly the photopic a- and b-waves and oscillatory potentials. Our study indicates that ASIC1a is an important channel in normal retinal activity. Being present in the inner segments of cones and inner nuclear layer cells, and mainly at synaptic cleft levels, it could participate in gain adaptation to ambient light of the cone pathway, facilitating cone hyperpolarization in brightness and modulating synaptic transmission of the light-induced visual signal.
Project description:We have previously postulated that unconventional myosin VI (MVI) could be involved in myoblast differentiation. Here, we addressed the mechanism(s) of its involvement using primary myoblast culture derived from the hindlimb muscles of Snell's waltzer mice, the natural MVI knockouts (MVI-KO). We observed that MVI-KO myotubes were formed faster than control heterozygous myoblasts (MVI-WT), with a three-fold increase in the number of myosac-like myotubes with centrally positioned nuclei. There were also changes in the levels of the myogenic transcription factors Pax7, MyoD and myogenin. This was accompanied by changes in the actin cytoskeleton and adhesive structure organization. We observed significant decreases in the levels of proteins involved in focal contact formation, such as talin and focal adhesion kinase (FAK). Interestingly, the levels of proteins involved in intercellular communication, M-cadherin and drebrin, were also affected. Furthermore, time-dependent alterations in the levels of the key proteins for myoblast membrane fusion, myomaker and myomerger, without effect on their cellular localization, were observed. Our data indicate that in the absence of MVI, the mechanisms controlling cytoskeleton organization, as well as myoblast adhesion and fusion, are dysregulated, leading to the formation of aberrant myotubes.
Project description:Myosin Ia (Myo1a), the most prominent plus-end directed motor and myosin VI (Myo6) the sole minus-end directed motor, together exert opposing tension between the microvillar (MV) actin core and the apical brush border (BB) membrane of the intestinal epithelial cell (IEC). Mice lacking Myo1a or Myo6 each exhibit a variety of defects in the tethering of the BB membrane to the actin cytoskeleton. Double mutant (DM) mice lacking both myosins revealed that all the defects observed in either the Myo1a KO or Snell's waltzer (sv/sv) Myo6 mutant mouse are absent. In isolated DM BBs, Myo1a crosslinks between MV membrane and MV actin core are absent but the gap (which is lost in Myo1a KO) between the MV core and membrane is maintained. Several myosins including Myo1c, d, and e and Myo5a are ectopically recruited to the BB. Consistent with the restoration of membrane tethering defects by one or more of these myosins, upward ATP-driven shedding of the BB membrane, which is blocked in the Myo1a KO, is restored in the DM BB. However, Myo1a or Myo6 dependent defects in expression of membrane proteins that traffic between the BB membrane and endosome (NaPi2b, NHE3, CFTR) are not restored. Compared to controls, Myo1a KO, sv/sv mice exhibit moderate and DM high levels of hypersensitivity to dextran sulfate sodium-induced colitis. Consistent with Myo1a and Myo6 playing critical roles in maintaining IEC integrity and response to injury, DM IECs exhibit increased numbers of apoptotic nuclei, above that reported for Myo1a KO.
Project description:OTX2 is a homeoprotein transcription factor expressed in photoreceptors and bipolar cells in the retina. OTX2, like many other homeoproteins, transfers between cells and exerts non-cell autonomous effects such as promoting the survival of retinal ganglion cells that do not express the protein. Here we used a genetic approach to target extracellular OTX2 in the retina by conditional expression of a secreted single-chain anti-OTX2 antibody. Compared with control mice, the expression of this antibody by parvalbumin-expressing neurons in the retina is followed by a reduction in visual acuity in 1-month-old mice with no alteration of the retinal structure or cell type number or aspect. The a-waves and b-waves measured by electroretinogram were also indistinguishable from those of control mice, suggesting no functional deficit of photoreceptors and bipolar cells. Mice expressing the OTX2-neutralizing antibody did show a significant doubling in the flicker amplitude and a reduction in oscillatory potential, consistent with a change in inner retinal function. Our results show that interfering in vivo with OTX2 non-cell autonomous activity in the postnatal retina leads to an alteration in inner retinal cell functions and causes a deficit in visual acuity.
Project description:To investigate retinal microstructure of patients affected with malattia leventinese (MLVT) and mutation in the EFEMP1 gene using high-resolution optical coherence tomography (OCT).Patients diagnosed with MLVT received a comprehensive eye exam, full-field and multifocal electroretinogram testing and imaging with a high-resolution Fourier domain OCT (Fd-OCT, UC Davis Medical Center, Davis, USA; axial resolution: 4.5 microm, acquisition speed: 9 frames s(-1), 1000 A scans s(-1)) combined with a flexible scanning head (Bioptigen Inc. Durham, NC, USA).Two related patients aged 30 and 60 years, with MLVT and identified c.R345W mutation in the EFEMP1 gene, were tested. Mother and daughter showed a variable phenotype with reduced vision function in the younger patient, whereas the mother had a 'form frustre'. Fd-OCT revealed extensive or focal sub-retinal pigment epithelium (RPE) deposits, separation of RPE and Bruch's membrane, and disruption of the photoreceptor outer and inner segment layers. No outer retinal changes were visible outside areas with sub-RPE deposits.Retinal structure in EFEMP1 retinal dystrophy is reflected by morphological changes within the RPE/Bruch's membrane complex with accumulation of sub-RPE material associated with disrupted photoreceptor integrity. The pattern of microstructural retinal abnormalities is similar but with a different extent in patients with variable phenotypes.
Project description:Purpose:The purpose of this paper was to discuss manifestations of primary mitochondrial dysfunctions and whether the retinal pigment epithelium or the photoreceptors are preferentially affected. Methods:A retrospective analysis was performed of patients with clinically and laboratory confirmed diagnoses of maternally inherited diabetes and deafness (MIDD) or Kearns-Sayre syndrome (KSS). Patients underwent full ophthalmic examination, full-field electroretinogram, and multimodal imaging studies, including short-wavelength autofluorescence, spectral domain-optical coherence tomography, and color fundus photography. Results:A total of five patients with MIDD and four patients with KSS were evaluated at two tertiary referral centers. Mean age at initial evaluation was 50.3 years old. Nascent outer retinal tubulations corresponding with faint foci of autofluorescence were observed in two patients with MIDD. Characteristic features of this cohort included a foveal sparing phenotype observed in 13 of 18 eyes (72%), global absence of intraretinal pigment migration, and preserved retinal function on full-field electroretinogram testing in 12 of 16 eyes (75%). One patient diagnosed with MIDD presented with an unusual pattern of atrophy surrounding the parapapillary region and one patient with KSS presented with an atypical choroideremia-like phenotype. Conclusions:MIDD and KSS are phenotypically heterogeneous disorders. Several features of disease suggest that primary mitochondrial dysfunction may first affect the retinal pigment epithelium followed by secondary photoreceptor loss. Similarities between primary mitochondrial degenerations and retinal disorders, such as age-related macular degeneration may suggest a primary role of mitochondria in the pathogenesis of these oligogenic disorders.