RESUMEN
Microtubules are essential for various cellular processes. The functional diversity of microtubules is attributed to the incorporation of various α- and ß-tubulin isotypes encoded by different genes. In this work, we investigated the functional role of ß4B-tubulin isotype (TUBB4B) in hearing and vision as mutations in TUBB4B are associated with sensorineural disease. Using a Tubb4b knockout mouse model, our findings demonstrate that TUBB4B is essential for hearing. Mice lacking TUBB4B are profoundly deaf due to defects in the inner and middle ear. Specifically, in the inner ear, the absence of TUBB4B lead to disorganized and reduced densities of microtubules in pillar cells, suggesting a critical role for TUBB4B in providing mechanical support for auditory transmission. In the middle ear, Tubb4b-/- mice exhibit motile cilia defects in epithelial cells, leading to the development of otitis media. However, Tubb4b deletion does not affect photoreceptor function or cause retinal degeneration. Intriguingly, ß6-tubulin levels increase in retinas lacking ß4B-tubulin isotype, suggesting a functional compensation mechanism. Our findings illustrate the essential roles of TUBB4B in hearing but not in vision in mice, highlighting the distinct functions of tubulin isotypes in different sensory systems.
Asunto(s)
Cilios , Ratones Noqueados , Tubulina (Proteína) , Animales , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Cilios/metabolismo , Ratones , Citoesqueleto/metabolismo , Cóclea/metabolismo , Microtúbulos/metabolismoRESUMEN
Microtubules, polymers of αß-tubulin heterodimers, are essential for various cellular processes. The incorporation of different tubulin isotypes, each encoded by distinct genes, is proposed to contribute to the functional diversity observed in microtubules. However, the functional roles of each tubulin isotype are not completely understood. In this study, we investigated the role of the ß4B-tubulin isotype (Tubb4b) in spermatogenesis, utilizing a Tubb4b knockout mouse model. We showed that ß4B-tubulin is expressed in the germ cells throughout spermatogenesis. ß4B-tubulin was localized to cytoplasmic microtubules, mitotic spindles, manchette, and axonemes of sperm flagella. We found that the absence of ß4B-tubulin resulted in male infertility and failure to produce sperm cells. Our findings demonstrate that a lack of ß4B-tubulin leads to defects in the initial stages of spermatogenesis. Specifically, ß4B-tubulin is needed for the expansion of differentiating spermatogonia, which is essential for the subsequent progression of spermatogenesis.
Asunto(s)
Diferenciación Celular , Ratones Noqueados , Microtúbulos , Espermatogénesis , Espermatogonias , Tubulina (Proteína) , Animales , Masculino , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Espermatogonias/metabolismo , Espermatogonias/citología , Espermatogénesis/genética , Ratones , Microtúbulos/metabolismo , Infertilidad Masculina/genética , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patologíaRESUMEN
Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the stability of a small set of proteins essential in various cellular pathways. Cytosolic HSP90 has two closely related paralogs: HSP90α and HSP90ß. Due to the structural and sequence similarities of cytosolic HSP90 paralogs, identifying the unique functions and substrates in the cell remains challenging. In this article, we assessed the role of HSP90α in the retina using a novel HSP90α murine knockout model. Our findings show that HSP90α is essential for rod photoreceptor function but was dispensable in cone photoreceptors. In the absence of HSP90α, photoreceptors developed normally. We observed rod dysfunction in HSP90α knockout at 2 months with the accumulation of vacuolar structures, apoptotic nuclei, and abnormalities in the outer segments. The decline in rod function was accompanied by progressive degeneration of rod photoreceptors that was complete at 6 months. The deterioration in cone function and health was a "bystander effect" that followed the degeneration of rods. Tandem mass tag proteomics showed that HSP90α regulates the expression levels of <1% of the retinal proteome. More importantly, HSP90α was vital in maintaining rod PDE6 and AIPL1 cochaperone levels in rod photoreceptor cells. Interestingly, cone PDE6 levels were unaffected. The robust expression of HSP90ß paralog in cones likely compensates for the loss of HSP90α. Overall, our study demonstrated the critical need for HSP90α chaperone in the maintenance of rod photoreceptors and showed potential substrates regulated by HSP90α in the retina.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Regulación Enzimológica de la Expresión Génica , Proteínas HSP90 de Choque Térmico , Células Fotorreceptoras Retinianas Bastones , Animales , Ratones , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Proteínas HSP90 de Choque Térmico/deficiencia , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Bastones/citología , Células Fotorreceptoras Retinianas Bastones/enzimología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Subunidades de Proteína , Supervivencia CelularRESUMEN
Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.
Asunto(s)
Oxígeno , Compuestos de Tritilo , Animales , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Interacciones Hidrofóbicas e Hidrofílicas , RatonesRESUMEN
Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.
Asunto(s)
Proteínas Portadoras/genética , Ciliopatías/genética , Infertilidad Masculina/genética , Proteínas de Transporte de Membrana/genética , Fotofobia/genética , Adulto , Animales , Cilios/patología , Ciliopatías/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Infertilidad Masculina/patología , Masculino , Ratones , Ratones Noqueados , Microtúbulos/metabolismo , Persona de Mediana Edad , Linaje , Fotofobia/patología , Motilidad Espermática/genética , Cola del Espermatozoide/patología , Espermatogénesis/genética , Síndrome , Factores de TranscripciónRESUMEN
Enzyme activities are well established biomarkers of many pathologies. Imaging enzyme activity directly in vivo may help to gain insight into the pathogenesis of various diseases but remains extremely challenging. In this communication, we report the use of EPR imaging (EPRI) in combination with a specially designed paramagnetic enzymatic substrate to map alkaline phosphatase activity with a high selectivity, thereby demonstrating the potential of EPRI to map enzyme activity.
Asunto(s)
Fosfatasa Alcalina/análisis , Espectroscopía de Resonancia por Spin del Electrón/métodos , Fosfatasa Alcalina/metabolismo , Línea Celular Tumoral , Pruebas de Enzimas/métodos , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Fosforilación , Especificidad por SustratoRESUMEN
The study has demonstrated a dual effect of nitric oxide on phenoloxidase (PO)-mediated DOPA oxidation and melanization process. NO generated at low rates proportionally increased in PO-mediated DOPA oxidation. Competitive PO inhibitor, phenylthiourea, resulted in significant inhibition of NO-mediated DOPA oxidation. Further analysis using fluorescent and EPR methods demonstrated that the effect of NO on DOPA oxidation is explained by oxidation of NO to NO2 at the active site of PO followed by oxidation of DOPA by NO2. On the contrary, the bolus addition of NO gas solution resulted in a significant decrease in observed PO activity. Similar dose-dependent effect of NO was observed for the insect's haemocytes quantified as percentage of melanized cells after treatment with nitric oxide. In conclusion, the results of the study suggest that NO may have a significant regulatory role on melanization process in invertebrates as well as in human and result in protective or damaging effects.