scholarly journals Microtubule stability, Golgi organization, and transport flux require dystonin-a2–MAP1B interaction

2012 ◽  
Vol 196 (6) ◽  
pp. 727-742 ◽  
Author(s):  
Scott D. Ryan ◽  
Kunal Bhanot ◽  
Andrew Ferrier ◽  
Yves De Repentigny ◽  
Alphonse Chu ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Function Analysis ◽  
Trichostatin A ◽  
Mutant Mice ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Microtubule Stability ◽  
Golgi Fragmentation ◽  
Golgi Organization ◽  
Transport Flux

Loss of function of dystonin cytoskeletal linker proteins causes neurodegeneration in dystonia musculorum (dt) mutant mice. Although much investigation has focused on understanding dt pathology, the diverse cellular functions of dystonin isoforms remain poorly characterized. In this paper, we highlight novel functions of the dystonin-a2 isoform in mediating microtubule (MT) stability, Golgi organization, and flux through the secretory pathway. Using dystonin mutant mice combined with isoform-specific loss-of-function analysis, we found dystonin-a2 bound to MT-associated protein 1B (MAP1B) in the centrosomal region, where it maintained MT acetylation. In dt neurons, absence of the MAP1B–dystonin-a2 interaction resulted in altered MAP1B perikaryal localization, leading to MT deacetylation and instability. Deacetylated MT accumulation resulted in Golgi fragmentation and prevented anterograde trafficking via motor proteins. Maintenance of MT acetylation through trichostatin A administration or MAP1B overexpression mitigated the observed defect. These cellular aberrations are apparent in prephenotype dorsal root ganglia and primary sensory neurons from dt mice, suggesting they are causal in the disorder.

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

2004 ◽  
Vol 287 (3) ◽  
pp. C580-C589 ◽  
Author(s):  
Van-Khue Ton ◽  
Rajini Rao
Keyword(s):  
Secretory Pathway ◽  
Function Analysis ◽  
Model Organism ◽  
Functional Expression ◽  
Missense Mutations ◽  
Heterologous Proteins ◽  
Loss Of Function ◽  
Yeast Saccharomyces Cerevisiae ◽  
Culturing Conditions ◽  
Mutant Phenotypes

The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca2+ homeostasis machinery, with particular emphasis on Ca2+-transporting ATPases. Putative Ca2+-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca2+ pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca2+ transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca2+,Mn2+-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease.

Mash1 and Ngn1 control distinct steps of determination and differentiation in the olfactory sensory neuron lineage

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1871-1880 ◽  
Author(s):  
Elise Cau ◽  
Simona Casarosa ◽  
François Guillemot
Keyword(s):  
Sensory Neuron ◽  
Sensory Neurons ◽  
Function Analysis ◽  
Olfactory Sensory Neuron ◽  
Mutant Mice ◽  
Olfactory Sensory Neurons ◽  
Null Mutant ◽  
Loss Of Function ◽  
Bhlh Genes ◽  
Null Mutant Mice

bHLH transcription factors are expressed sequentially during the development of neural lineages, suggesting that they operate in genetic cascades. In the olfactory epithelium, the proneural genes Mash1 and neurogenin1 are expressed at distinct steps in the same olfactory sensory neuron lineage. Here, we show by loss-of-function analysis that both genes are required for the generation of olfactory sensory neurons. However, their mutant phenotypes are strikingly different, indicating that they have divergent functions. In Mash1 null mutant mice, olfactory progenitors are not produced and the Notch signalling pathway is not activated, establishing Mash1 as a determination gene for olfactory sensory neurons. In neurogenin1 null mutant mice, olfactory progenitors are generated but they express only a subset of their normal repertoire of regulatory molecules and their differentiation is blocked. Thus neurogenin1 is required for the activation of one of several parallel genetic programs functioning downstream of Mash1 in the differentiation of olfactory sensory neurons. These results illustrate the versatility of neural bHLH genes which adopt either a determination or a differentiation function, depending primarily on the timing of their expression in neural progenitors.

scholarly journals The molecular chaperone Hsp90 maintains Golgi organization and vesicular trafficking by regulating microtubule stability

2019 ◽  
Vol 12 (6) ◽  
pp. 448-461 ◽  
Author(s):  
Yuan Wu ◽  
Yubo Ding ◽  
Xiudan Zheng ◽  
Kan Liao
Keyword(s):  
Transcription Factors ◽  
Golgi Apparatus ◽  
Molecular Chaperone ◽  
Trichostatin A ◽  
Vesicular Trafficking ◽  
High Abundance ◽  
Microtubule Stabilization ◽  
Cellular Event ◽  
Microtubule Stability ◽  
Golgi Organization

Abstract Hsp90 is an abundant and special molecular chaperone considered to be the regulator of many transcription factors and signaling kinases. Its high abundance is indicative of its involvement in some more fundamental processes. In this study, we provide evidence that Hsp90 is required for microtubule stabilization, Golgi organization, and vesicular trafficking. We showed that Hsp90 is bound to microtubule-associated protein 4 (MAP4), which is essential for maintaining microtubule acetylation and stabilization. Hsp90 depletion led to the decrease in MAP4, causing microtubule deacetylation and destabilization. Furthermore, in Hsp90-depleted cells, the Golgi apparatus was fragmented and anterograde vesicle trafficking was impaired, with phenotypes similar to those induced by silencing MAP4. These disruptive effects of Hsp90 depletion could be rescued by the expression of exogenous MAP4 or the treatment of trichostatin A that increases microtubule acetylation as well as stability. Thus, microtubule stability is an essential cellular event regulated by Hsp90.

scholarly journals Mask, the Drosophila Ankyrin Repeat and KH domain-containing protein, affects microtubule stability

2021 ◽  
Author(s):  
Daniel Martinez ◽  
Mingwei Zhu ◽  
Jessie J. Guidry ◽  
Niles Majeste ◽  
Hui Mao ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neuromuscular Junctions ◽  
Function Analysis ◽  
Normal Function ◽  
Loss Of Function ◽  
Synaptic Growth ◽  
Cellular Processes ◽  
Microtubule Stability ◽  
Kh Domain ◽  
Mask Function

Proper regulation of microtubule (MT) stability and dynamics is vital for essential cellular processes, including axonal transportation and synaptic growth and remodeling in neurons. Here, we demonstrate that Mask negatively affects MT stability in both fly larval muscles and motor neurons. In larval muscles, loss-of-function of mask increases MT polymer length, and in motor neurons, loss of mask function results in overexpansion of the presynaptic terminal at the larval neuromuscular junctions (NMJs). mask genetically interacts with stathmin (stai), a neuronal modulator of MT stability, in the regulation of axon transportation and synaptic terminal stability. Our structure/function analysis on Mask revealed that its Ankyrin Repeats domain-containing N-terminal portion is sufficient to mediate Mask's impact to MT stability. Furthermore, we discovered that Mask negatively regulates the abundance of the microtubule-associated protein Jupiter in motor neuron axons, and that neuronal knocking down of Jupiter partially suppresses mask loss-of-function phenotypes at the larval NMJs. Together, our studies demonstrated that Mask is a novel regulator for microtubule stability, and such a role of Mask requires normal function of Jupiter.

scholarly journals ATP7A-Regulated Enzyme Metalation and Trafficking in the Menkes Disease Puzzle

Biomedicines ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 391
Author(s):  
Nina Horn ◽  
Pernilla Wittung-Stafshede
Keyword(s):  
Secretory Pathway ◽  
Current Knowledge ◽  
Lysyl Oxidase ◽  
Menkes Disease ◽  
The Body ◽  
Whole Body ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Inborn Errors ◽  
Organ Systems

Copper is vital for numerous cellular functions affecting all tissues and organ systems in the body. The copper pump, ATP7A is critical for whole-body, cellular, and subcellular copper homeostasis, and dysfunction due to genetic defects results in Menkes disease. ATP7A dysfunction leads to copper deficiency in nervous tissue, liver, and blood but accumulation in other tissues. Site-specific cellular deficiencies of copper lead to loss of function of copper-dependent enzymes in all tissues, and the range of Menkes disease pathologies observed can now be explained in full by lack of specific copper enzymes. New pathways involving copper activated lysosomal and steroid sulfatases link patient symptoms usually related to other inborn errors of metabolism to Menkes disease. Additionally, new roles for lysyl oxidase in activation of molecules necessary for the innate immune system, and novel adapter molecules that play roles in ERGIC trafficking of brain receptors and other proteins, are emerging. We here summarize the current knowledge of the roles of copper enzyme function in Menkes disease, with a focus on ATP7A-mediated enzyme metalation in the secretory pathway. By establishing mechanistic relationships between copper-dependent cellular processes and Menkes disease symptoms in patients will not only increase understanding of copper biology but will also allow for the identification of an expanding range of copper-dependent enzymes and pathways. This will raise awareness of rare patient symptoms, and thus aid in early diagnosis of Menkes disease patients.

scholarly journals Roles for RNA export factor, Nxt1, in ensuring muscle integrity and normal RNA expression in Drosophila

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin van der Graaf ◽  
Katia Jindrich ◽  
Robert Mitchell ◽  
Helen White-Cooper
Keyword(s):  
Mrna Export ◽  
Rna Stability ◽  
Muscle Degeneration ◽  
Loss Of Function ◽  
Cellular Functions ◽  
Partial Loss ◽  
Export Pathway ◽  
Pathway Gene ◽  
Rna Export

Abstract The mRNA export pathway is responsible for the transport of mRNAs from the nucleus to the cytoplasm, and thus is essential for protein production and normal cellular functions. A partial loss of function allele of the mRNA export factor Nxt1 in Drosophila shows reduced viability and sterility. A previous study has shown that the male fertility defect is due to a defect in transcription and RNA stability, indicating the potential for this pathway to be implicated in processes beyond the known mRNA transport function. Here we investigate the reduced viability of Nxt1 partial loss of function mutants, and describe a defect in growth and maintenance of the larval muscles, leading to muscle degeneration. RNA-seq revealed reduced expression of a set of mRNAs, particularly from genes with long introns in Nxt1 mutant carcass. We detected differential expression of circRNA, and significantly fewer distinct circRNAs expressed in the mutants. Despite the widespread defects in gene expression, muscle degeneration was rescued by increased expression of the costamere component tn (abba) in muscles. This is the first report of a role for the RNA export pathway gene Nxt1 in the maintenance of muscle integrity. Our data also links the mRNA export pathway to a specific role in the expression of mRNA and circRNA from common precursor genes, in vivo.

Genetic analysis of Pycr1 and Pycr2 in mice

Genetics ◽  
2021 ◽  
Author(s):  
Morgane G Stum ◽  
Abigail L D Tadenev ◽  
Kevin L Seburn ◽  
Kathy E Miers ◽  
Pak P Poon ◽  
...  
Keyword(s):  
Neuromuscular Disorders ◽  
Subcutaneous Fat ◽  
Mutant Mice ◽  
Loss Of Function ◽  
Proline Biosynthesis ◽  
Cell Counts ◽  
Chemically Induced ◽  
White Blood Cell Counts ◽  
Redundant Functions ◽  
Altered Lipid

Abstract The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.

scholarly journals HMGB1 Promotes Systemic Lupus Erythematosus by Enhancing Macrophage Inflammatory Response

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Mudan Lu ◽  
Shanshan Yu ◽  
Wei Xu ◽  
Bo Gao ◽  
Sidong Xiong
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Inflammatory Response ◽  
Proinflammatory Cytokines ◽  
Lupus Erythematosus ◽  
Function Analysis ◽  
Critical Role ◽  
Loss Of Function ◽  
Systemic Lupus ◽  
Glycation End Products ◽  
Hmgb1 Expression

Background/Purpose. HMGB1, which may act as a proinflammatory mediator, has been proposed to contribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases including systemic lupus erythematosus (SLE); however, the precise mechanism of HMGB1 in the pathogenic process of SLE remains obscure.Method. The expression of HMGB1 was measured by ELISA and western blot. The ELISA was also applied to detect proinflammatory cytokines levels. Furthermore, nephritic pathology was evaluated by H&E staining of renal tissues.Results. In this study, we found that HMGB1 levels were significantly increased and correlated with SLE disease activity in both clinical patients and murine model. Furthermore, gain- and loss-of-function analysis showed that HMGB1 exacerbated the severity of SLE. Of note, the HMGB1 levels were found to be associated with the levels of proinflammatory cytokines such as TNF-αand IL-6 in SLE patients. Further study demonstrated that increased HMGB1 expression deteriorated the severity of SLE via enhancing macrophage inflammatory response. Moreover, we found that receptor of advanced glycation end products played a critical role in HMGB1-mediated macrophage inflammatory response.Conclusion. These findings suggested that HMGB1 might be a risk factor for SLE, and manipulation of HMGB1 signaling might provide a therapeutic strategy for SLE.

scholarly journals Molecular mechanism of ATP versus GTP selectivity of adenylate kinase

2018 ◽  
Vol 115 (12) ◽  
pp. 3012-3017 ◽  
Author(s):  
Per Rogne ◽  
Marie Rosselin ◽  
Christin Grundström ◽  
Christian Hedberg ◽  
Uwe H. Sauer ◽  
...  
Keyword(s):  
Hydrogen Bond ◽  
Adenylate Kinase ◽  
Function Analysis ◽  
Substrate Selectivity ◽  
Cellular Functions ◽  
X Ray ◽  
Precise Control ◽  
X Ray Crystallography ◽  
The Family ◽  
Atp Analogs

Enzymatic substrate selectivity is critical for the precise control of metabolic pathways. In cases where chemically related substrates are present inside cells, robust mechanisms of substrate selectivity are required. Here, we report the mechanism utilized for catalytic ATP versus GTP selectivity during adenylate kinase (Adk) -mediated phosphorylation of AMP. Using NMR spectroscopy we found that while Adk adopts a catalytically competent and closed structural state in complex with ATP, the enzyme is arrested in a catalytically inhibited and open state in complex with GTP. X-ray crystallography experiments revealed that the interaction interfaces supporting ATP and GTP recognition, in part, are mediated by coinciding residues. The mechanism provides an atomic view on how the cellular GTP pool is protected from Adk turnover, which is important because GTP has many specialized cellular functions. In further support of this mechanism, a structure–function analysis enabled by synthesis of ATP analogs suggests that a hydrogen bond between the adenine moiety and the backbone of the enzyme is vital for ATP selectivity. The importance of the hydrogen bond for substrate selectivity is likely general given the conservation of its location and orientation across the family of eukaryotic protein kinases.

scholarly journals MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

2021 ◽  
Author(s):  
Janine McCaughey ◽  
Judith M. Mantell ◽  
Chris R. Neal ◽  
Kate Heesom ◽  
David J. Stephens
Keyword(s):  
Endoplasmic Reticulum ◽  
Light Microscopy ◽  
Secretory Pathway ◽  
Genome Engineering ◽  
High Volume ◽  
Vesicle Budding ◽  
Early Secretory Pathway ◽  
Copii Vesicle ◽  
Intermediate Compartment ◽  
Golgi Organization

AbstractComplex machinery is required to drive secretory cargo export from the endoplasmic reticulum. In vertebrates, this includes transport and Golgi organization protein 1 (TANGO1), encoded by the Mia3 gene. Here, using genome engineering of human cells light microscopy, secretion assays, and proteomics, we show loss of Mia3/TANGO1 results in formation of numerous vesicles and a loss of early secretory pathway integrity. This restricts secretion not only of large proteins like procollagens but of all types of secretory cargo. Our data shows that Mia3/TANGO1 constrains the propensity of COPII to form vesicles promoting instead the formation of the ER-Golgi intermediate compartment. Thus, Mia3/TANGO1 facilities the secretion of complex and high volume cargoes from vertebrate cells.

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