Tight Functional Coupling of Kinesin-1A and Dynein Motors in the Bidirectional Transport of Neurofilaments

We have tested the hypothesis that kinesin-1A (formerly KIF5A) is an anterograde motor for axonal neurofilaments. In cultured sympathetic neurons from kinesin-1A knockout mice, we observed a 75% reduction in the frequency of both anterograde and retrograde neurofilament movement. This transport defect could be rescued by kinesin-1A, and with successively decreasing efficacy by kinesin-1B and kinesin-1C. In wild-type neurons, headless mutants of kinesin-1A and kinesin-1C inhibited both anterograde and retrograde movement in a dominant-negative manner. Because dynein is thought to be the retrograde motor for axonal neurofilaments, we investigated the effect of dynein inhibition on anterograde and retrograde neurofilament transport. Disruption of dynein function by using RNA interference, dominant-negative approaches, or a function-blocking antibody also inhibited both anterograde and retrograde neurofilament movement. These data suggest that kinesin-1A is the principal but not exclusive anterograde motor for neurofilaments in these neurons, that there may be some functional redundancy among the kinesin-1 isoforms with respect to neurofilament transport, and that the activities of the anterograde and retrograde neurofilament motors are tightly coordinated.

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Association of distinct type 1 bone morphogenetic protein receptors with different molecular pathways and survival outcomes in neuroblastoma

Neuronal Signaling ◽

10.1042/ns20200006 ◽

2020 ◽

Vol 4 (1) ◽

Author(s):

Amnah M. Alshangiti ◽

Sean L. Wyatt ◽

Erin McCarthy ◽

Louise M. Collins ◽

Shane V. Hegarty ◽

...

Keyword(s):

Bone Morphogenetic Protein ◽

Bone Morphogenetic Proteins ◽

Sympathetic Neurons ◽

Distinct Type ◽

Dominant Negative ◽

Survival Outcomes ◽

Mycn Amplification ◽

Morphogenetic Protein ◽

Stage 4 ◽

Cultured Sympathetic Neurons

Abstract Neuroblastoma (NB) is a paediatric cancer that arises in the sympathetic nervous system. Patients with stage 4 tumours have poor outcomes and 20% of high-risk cases have MYCN amplification. The bone morphogenetic proteins (BMPs) play roles in sympathetic neuritogenesis, by signalling through bone morphogenetic protein receptor (BMPR)2 and either BMPR1A or BMPR1B. Alterations in BMPR2 expression have been reported in NB; it is unknown if the expression of BMPR1A or BMPR1B is altered. We report lower BMPR2 and BMPR1B, and higher BMPR1A, expression in stage 4 and in MYCN-amplified NB. Kaplan–Meier plots showed that high BMPR2 or BMPR1B expression was linked to better survival, while high BMPR1A was linked to worse survival. Gene ontology enrichment and pathway analyses revealed that BMPR2 and BMPR1B co-expressed genes were enriched in those associated with NB differentiation. BMPR1A co-expressed genes were enriched in those associated with cell proliferation. Moreover, the correlation between BMPR2 and BMPR1A was strengthened, while the correlation between BMPR2 and BMPR1B was lost, in MYCN-amplified NB. This suggested that differentiation should decrease BMPR1A and increase BMPR1B expression. In agreement, nerve growth factor treatment of cultured sympathetic neurons decreased Bmpr1a expression and increased Bmpr1b expression. Overexpression of dominant negative BMPR1B, treatment with a BMPR1B inhibitor and treatment with GDF5, which signals via BMPR1B, showed that BMPR1B signalling is required for optimal neuritogenesis in NB cells, suggesting that loss of BMPR1B may alter neuritogenesis. The present study shows that expression of distinct BMPRs is associated with different survival outcomes in NB.

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Rescue of Pituitary Function in a Mouse Model of Isolated Growth Hormone Deficiency Type II by RNA Interference

Endocrinology ◽

10.1210/en.2007-1360 ◽

2007 ◽

Vol 149 (2) ◽

pp. 580-586 ◽

Cited By ~ 11

Author(s):

Nikki Shariat ◽

Robin C. C. Ryther ◽

John A. Phillips ◽

Iain C. A. F. Robinson ◽

James G. Patton

Keyword(s):

Rna Interference ◽

Gh Deficiency ◽

Dominant Negative ◽

Hormone Deficiency ◽

Type Ii ◽

Wild Type ◽

Pituitary Hypoplasia ◽

Short Hairpin ◽

Deficiency Type

Splicing mutations in the human GH (hGH) gene (GH-1) that cause skipping of exon 3 result in a form of GH deficiency termed isolated GH deficiency type II (IGHD II). The GH-1 gene contains five exons; constitutive splicing produces the wild-type 22-kDa hormone, whereas skipping of exon 3 results in transcripts encoding a 17.5-kDa isoform that acts as a dominant-negative to block secretion of the wild-type hormone. Common characteristics of IGHD II include short stature due to impaired bone elongation, growth, and, in severe cases, anterior pituitary hypoplasia. Typically, IGHD II is treated by sc delivery of hGH, which can rescue stature but, unfortunately, does not inhibit pituitary hypoplasia. Direct destruction of transcripts encoding the dominant-negative 17.5-kDa isoform should both rescue stature and prevent hypoplasia. Here, we have used delivery of short hairpin RNAs to rescue a murine model of IGHD II by specifically targeting transcripts encoding the 17.5-kDa isoform using RNA interference. To our knowledge, this is the first example where a short hairpin RNA has been expressed to specifically degrade an incorrectly spliced transcript and rescue a dominant-negative disease phenotype in vivo.

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The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽

10.3390/biom11050651 ◽

2021 ◽

Vol 11 (5) ◽

pp. 651

Author(s):

Hsiao-Cheng Tsai ◽

Che-Hong Chen ◽

Daria Mochly-Rosen ◽

Yi-Chen Ethan Li ◽

Min-Huey Chen

Keyword(s):

Bone Loss ◽

Bacterial Infection ◽

Bone Regeneration ◽

Alcohol Drinking ◽

Bacterial Species ◽

Dominant Negative ◽

Enzyme Inactivation ◽

Wild Type ◽

Micro Computed Tomography ◽

Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

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Analysis of maxillopedia Expression Pattern and Larval Cuticular Phenotype in Wild-Type and Mutant Tribolium

Genetics ◽

10.1093/genetics/155.2.721 ◽

2000 ◽

Vol 155 (2) ◽

pp. 721-731 ◽

Cited By ~ 7

Author(s):

Teresa D Shippy ◽

Jianhua Guo ◽

Susan J Brown ◽

Richard W Beeman ◽

Robin E Denell

Keyword(s):

Rna Interference ◽

Expression Pattern ◽

Tribolium Castaneum ◽

Expression Patterns ◽

Ectopic Expression ◽

Homeotic Gene ◽

Wild Type ◽

Double Stranded Rna ◽

Similar Expression ◽

Mutant Phenotypes

Abstract The Tribolium castaneum homeotic gene maxillopedia (mxp) is the ortholog of Drosophila proboscipedia (pb). Here we describe and classify available mxp alleles. Larvae lacking all mxp function die soon after hatching, exhibiting strong transformations of maxillary and labial palps to legs. Hypomorphic mxp alleles produce less severe transformations to leg. RNA interference with maxillopedia double-stranded RNA results in phenocopies of mxp mutant phenotypes ranging from partial to complete transformations. A number of gain-of-function (GOF) mxp alleles have been isolated based on transformations of adult antennae and/or legs toward palps. Finally, we have characterized the mxp expression pattern in wild-type and mutant embryos. In normal embryos, mxp is expressed in the maxillary and labial segments, whereas ectopic expression is observed in some GOF variants. Although mxp and Pb display very similar expression patterns, pb null embryos develop normally. The mxp mutant larval phenotype in Tribolium is consistent with the hypothesis that an ancestral pb-like gene had an embryonic function that was lost in the lineage leading to Drosophila.

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A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium

The Journal of Cell Biology ◽

10.1083/jcb.200611056 ◽

2007 ◽

Vol 176 (3) ◽

pp. 263-268 ◽

Cited By ~ 107

Author(s):

Adam C. Smith ◽

Won Do Heo ◽

Virginie Braun ◽

Xiuju Jiang ◽

Chloe Macrae ◽

...

Keyword(s):

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Dominant Negative ◽

Rab Gtpases ◽

Wild Type ◽

Intracellular Growth ◽

Non Invasive ◽

Serovar Typhimurium ◽

Guanosine Triphosphatase ◽

Kinetics Of

Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome–lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.

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Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and β1-integrin-dependent mechanism

AJP Cell Physiology ◽

10.1152/ajpcell.00543.2009 ◽

2010 ◽

Vol 299 (1) ◽

pp. C21-C32 ◽

Cited By ~ 11

Author(s):

Michael P. Hunter ◽

Mirjam M. Zegers

Keyword(s):

Cell Culture ◽

Mdck Cell ◽

Three Dimensional ◽

3D Culture ◽

Branching Morphogenesis ◽

Dominant Negative ◽

Β1 Integrin ◽

Blocking Antibody ◽

Important Regulator ◽

P21 Activated Kinase

Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-ΔPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a β1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and β1-integrin signaling.

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A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5

Blood ◽

10.1182/blood-2006-05-025221 ◽

2006 ◽

Vol 109 (8) ◽

pp. 3417-3423 ◽

Cited By ~ 55

Author(s):

Marina Bousquet ◽

Cyril Broccardo ◽

Cathy Quelen ◽

Fabienne Meggetto ◽

Emilienne Kuhlein ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Quantitative Polymerase Chain Reaction ◽

Dominant Negative ◽

Leukemic Cells ◽

Dominant Negative Effect ◽

Wild Type ◽

Cell Acute Lymphoblastic Leukemia

Abstract We report a novel t(7;9)(q11;p13) translocation in 2 patients with B-cell acute lymphoblastic leukemia (B-ALL). By fluorescent in situ hybridization and 3′ rapid amplification of cDNA ends, we showed that the paired box domain of PAX5 was fused with the elastin (ELN) gene. After cloning the full-length cDNA of the chimeric gene, confocal microscopy of transfected NIH3T3 cells and Burkitt lymphoma cells (DG75) demonstrated that PAX5-ELN was localized in the nucleus. Chromatin immunoprecipitation clearly indicated that PAX5-ELN retained the capability to bind CD19 and BLK promoter sequences. To analyze the functions of the chimeric protein, HeLa cells were cotransfected with a luc-CD19 construct, pcDNA3-PAX5, and with increasing amounts of pcDNA3-PAX5-ELN. Thus, in vitro, PAX5-ELN was able to block CD19 transcription. Furthermore, real-time quantitative polymerase chain reaction (RQ-PCR) experiments showed that PAX5-ELN was able to affect the transcription of endogenous PAX5 target genes. Since PAX5 is essential for B-cell differentiation, this translocation may account for the blockage of leukemic cells at the pre–B-cell stage. The mechanism involved in this process appears to be, at least in part, through a dominant-negative effect of PAX5-ELN on the wild-type PAX5 in a setting ofPAX5 haploinsufficiency.

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RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells

Neurochemical Research ◽

10.1007/s11064-010-0177-4 ◽

2010 ◽

Vol 35 (8) ◽

pp. 1214-1223 ◽

Cited By ~ 5

Author(s):

Xue-Ping Chen ◽

Xiao-Hui Hu ◽

Shu-Hui Wu ◽

Yang-Wei Zhang ◽

Bo Xiao ◽

...

Keyword(s):

Oxidative Stress ◽

Rna Interference ◽

Cultured Cells ◽

Wild Type

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Differential targeting and function of α2A and α2C adrenergic receptor subtypes in cultured sympathetic neurons

Neuropharmacology ◽

10.1016/j.neuropharm.2006.03.032 ◽

2006 ◽

Vol 51 (3) ◽

pp. 397-413 ◽

Cited By ~ 26

Author(s):

Patricia C. Brum ◽

Carl M. Hurt ◽

Olga G. Shcherbakova ◽

Brian Kobilka ◽

Timothy Angelotti

Keyword(s):

Adrenergic Receptor ◽

Sympathetic Neurons ◽

Receptor Subtypes ◽

Cultured Sympathetic Neurons ◽

And Function

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Suppressor Mutations Bypass the Requirement of fluG for Asexual Sporulation and Sterigmatocystin Production in Aspergillus nidulans

Genetics ◽

10.1093/genetics/165.3.1083 ◽

2003 ◽

Vol 165 (3) ◽

pp. 1083-1093

Author(s):

Jeong-Ah Seo ◽

Yajun Guan ◽

Jae-Hyuk Yu

Keyword(s):

Aspergillus Nidulans ◽

Molecular Mechanisms ◽

Dominant Negative ◽

Wild Type ◽

Dominant Mutation ◽

Site Mutation ◽

Asexual Sporulation ◽

Dominant Negative Mutation ◽

Suppressor Mutations ◽

Early Developmental

Abstract Asexual sporulation (conidiation) in the filamentous fungus Aspergillus nidulans requires the early developmental activator fluG. Loss of fluG results in the blockage of both conidiation and production of the mycotoxin sterigmatocystin (ST). To investigate molecular mechanisms of fluG-dependent developmental activation, 40 suppressors of fluG (SFGs) that conidiate without fluG have been isolated and characterized. Genetic analyses showed that an individual suppression is caused by a single second-site mutation, and that all sfg mutations but one are recessive. Pairwise meiotic crosses grouped mutations to four loci, 31 of them to sfgA, 6 of them to sfgB, and 1 each to sfgC and sfgD, respectively. The only dominant mutation, sfgA38, also mapped to the sfgA locus, suggesting a dominant negative mutation. Thirteen sfgA and 1 sfgC mutants elaborated conidiophores in liquid submerged culture, indicating that loss of either of these gene functions not only bypasses fluG function but also results in hyperactive conidiation. While sfg mutants show varying levels of restored conidiation, all recovered the ability to produce ST at near wild-type levels. The fact that at least four loci are defined by recessive sfg mutations indicates that multiple genes negatively regulate conidiation downstream of fluG and that the activity of fluG is required to remove such repressive effects.

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