Identification of a Novel Link between the Motor Proteins Dynein and Kinesin-1

Title from PDF of title page, viewed on August 30, 2016Dissertation advisor: Michael D. PlamannVitaIncludes bibliographical references (pages 93-115)Thesis (Ph.D.)--School of Biological Sciences. University of Missouri--Kansas City, 2016The motor proteins dynein and kinesin are fascinating biological machines which, like vehicles in a city, move various cellular cargoes along cytoskeletal microtubules (MT). In filamentous fungi, these motors are important for the hyphal growth, which is characterized by localized extension at the tip of the apical hyphal cell. This type of growth requires the movement of a large number of vesicles from and to the hyphal tip in order to support the continuous addition of new plasma membrane and cell wall. Recent studies in filamentous fungi, have shown that early endosomes (EEs) are specifically recognized and moved by dynein from the MT plus-ends at the hyphal tips to more distal cellular compartments. Interestingly, the targeting of dynein to MT plus-ends, is dependent on kinesin-1, and its disruption lead to an aberrant accumulation of EEs at hyphal tips and a reduction in mycelial expansion. While great advances have been made in our understanding of dynein interaction with its specific cargoes, the mechanism of its interaction with kinesin-1 have remained unknown. In an effort to expand our understanding of dynein physical and regulatory interactions, we employed genetic, molecular, and fluorescence microscopy techniques to isolate and analyze mutants affected in hyphal growth and in the localization of cytoplasmic dynein in the model organism Neurosporacrassa. Here we report the identification of a novel protein, which is required for the physical interaction between dynein and kinesin-1 during their journey to the hyphal tip. Interestingly, this protein is only detected in the genomes of the filamentous Ascomycota species but have the ability to physically interact with Drosophila kinesin-1, DmKHC.Introduction -- Materials and methods -- ROPY-16, a novel link between dynein and kinesin-1 -- Conclusio

TILLING to detect induced mutations in soybean

<p>Abstract</p> <p>Background</p> <p>Soybean (<it>Glycine max </it>L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).</p> <p>Results</p> <p>We screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.</p> <p>Conclusion</p> <p>The development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.</p

Neodiversification of homeologous CLAVATA1-like receptor kinase genes in soybean leads to distinct developmental outcomes

The CLAVATA pathway that regulates stem cell numbers of the shoot apical meristem has exclusively been studied in Arabidopsis; as such insight into other species is warranted. In this study, a GmCLV1A mutant (F-S562L) with altered lateral organ development, and two mutants of GmNARK, isolated from a Forrest M2 population (EMS-mutated soybean) were studied. GmCLV1A and GmNARK encode for LRR receptor kinases, and share 92% of protein sequence. While GmNARK is critical for systemic regulation of nodulation (new organ made on the root through symbiosis), we show that GmCLV1A functions locally and has no apparent function in nodulation or root development. However, a recessive, loss-offunction mutation (S562L) in a putative S-glycosylation site of GmCLV1A causes stem nodal identity alterations as well as flower and pod abnormalities (deformed flower and pod). The mutant also exhibits a homeotic phenotype, displaying abnormal leaf development/number, vein-derived leaf emergence, and a thick, faciated stem. The mutant phenotype is also temperature-sensitive. Interestingly, a novel truncated version of GmCLV1A was identified upstream of GmCLV1A that is absent from GmNARK, but is present upstream of the GmNARK orthologues, MtSUNN and PvNARK. Taken together, our findings indicate that GmCLV1A acts on shoot architecture, whereas GmNARK, functions in controlling nodule numbers

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts

Characterization of a Novel Prevacuolar Compartment in Neurospora crassa

Using confocal microscopy, we observed ring-like organelles, similar in size to nuclei, in the hyphal tip of the filamentous fungus Neurospora crassa. These organelles contained a subset of vacuolar proteins. We hypothesize that they are novel prevacuolar compartments (PVCs). We examined the locations of several vacuolar enzymes and of fluorescent compounds that target the vacuole. Vacuolar membrane proteins, such as the vacuolar ATPase (VMA-1) and the polyphosphate polymerase (VTC-4), were observed in the PVCs. A pigment produced by adenine auxotrophs, used to visualize vacuoles, also accumulated in PVCs. Soluble enzymes of the vacuolar lumen, alkaline phosphatase and carboxypeptidase Y, were not observed in PVCs. The fluorescent molecule Oregon Green 488 carboxylic acid diacetate, succinimidyl ester (carboxy-DFFDA) accumulated in vacuoles and in a subset of PVCs, suggesting maturation of PVCs from the tip to distal regions. Three of the nine Rab GTPases in N. crassa, RAB-2, RAB-4, and RAB-7, localized to the PVCs. RAB-2 and RAB-4, which have similar amino acid sequences, are present in filamentous fungi but not in yeasts, and no function has previously been reported for these Rab GTPases in fungi. PVCs are highly pleomorphic, producing tubular projections that subsequently become detached. Dynein and dynactin formed globular clusters enclosed inside the lumen of PVCs. The size, structure, dynamic behavior, and protein composition of the PVCs appear to be significantly different from those of the well-studied prevacuolar compartment of yeasts

TILLING to detect induced mutations in soybean.

BackgroundSoybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the world's protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).ResultsWe screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (approximately 1/140 kb), while another EMS population had a mutation density of approximately 1/250 kb. The remaining population had a mutation density of approximately 1/550 kb. Because of soybean's polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.ConclusionThe development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome

Type and distribution of induced mutations discovered in seven amplicons

Orange boxes correspond to exons, lines to introns. Homology to proteins in the BLOCKS database [38] is indicated by the green boxes above gmppck4 and gmrhg4b. The other amplicons did not contain regions of BLOCKS homology. Arrowheads indicate approximate position of missense changes, upside down arrowheads indicate silent changes, asterisks indicate nonsense mutations, boxes indicate deletions. Hollow arrowheads = A population; red = B population; gray = C population; black = D population. The number of mutations discovered in each amplicon per population is indicated on the right.<p><b>Copyright information:</b></p><p>Taken from "TILLING to detect induced mutations in soybean"</p><p>http://www.biomedcentral.com/1471-2229/8/9</p><p>BMC Plant Biology 2008;8():9-9.</p><p>Published online 24 Jan 2008</p><p>PMCID:PMC2266751.</p><p></p