Mingang Li

Differential expression of six glutamine synthetase genes in Zea mays

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3′ gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

Root- and shoot-specific responses of individual glutamine synthetase genes of maize to nitrate and ammonium

The responses of the five cytosolic-type glutamine synthetase (GS1) genes of maize to treatment of hydroponically grown seedlings with 10 mM KNO3 or 10 mM NH4Cl were analyzed. Non-coding 3′ gene-specific hybridization probes and radioanalytic imaging were used to quantitate individual gene transcript levels in excised roots and shoots before treatment and at selected times after treatment. Genes GS1−1 and GS1−2 exhibited distinct organ-specific responses to treatment with either nitrogen source. The GS1−1 transcript level increased over three-fold in roots, but changed little if any in shoots. In contrast, the GS1−2 transcript level increased over two-fold in shoots, but decreased in roots after treatment. Increased transcript levels were evident at 4 h after treatment with either nitrogen source, with maximum accumulations present at 8 h after treatment with ammonium and at 10–12 h after treatment with nitrate. The GS1−3 gene transcript level showed little or no change after treatment with either nitrogen source. The GS1−4 gene transcript level remained constant in shoots of treated seedlings, whereas in roots, it exhibited relatively minor, but complex responses to these two nitrogen sources. The GS1−5 gene transcript is present in very small amounts in seedlings, making it difficult to analyze its response to metabolites in young plants. These results provide support for the possibility that different cytosolic GS genes of maize play distinct roles in nitrogen metabolism during plant growth and differentiation.

Dynein Light Intermediate Chain: An Essential Subunit That Contributes to Spindle Checkpoint Inactivation

The dynein light intermediate chain (LIC) is a subunit unique to the cytoplasmic form of dynein, but how it contributes to dynein function is not fully understood. Previous work has established that the LIC homodimer binds directly to the dynein heavy chain and may mediate the attachment of dynein to centrosomes and other cargoes. Here, we report our characterization of the LIC in Drosophila. Unlike vertebrates, in which two Lic genes encode multiple subunit isoforms, the Drosophila LIC is encoded by a single gene. We determined that the single LIC polypeptide is phosphorylated, and that different phosphoisoforms can assemble into the dynein motor complex. Our mutational analyses demonstrate that, similar to other dynein subunits, the Drosophila LIC is required for zygotic development, germline specification of the oocyte, and mitotic cell division. We show that RNA interference depletion of LIC in Drosophila S2 cells does not block the recruitment of a dynein complex to kinetochores, but it does delay inactivation of Mad2 signaling and mitotic progression. Our observations suggest the LIC contributes to a broad range of dynein functions.

Motility Screen Identifies Drosophila IGF-II mRNA-Binding Protein—Zipcode-Binding Protein Acting in Oogenesis and Synaptogenesis

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, we have limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, we identified the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, we establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. We report here that loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. Our data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, we implicate a specific Imp domain in the establishment of dorsoventral polarity.

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.

Dynein and star interact in EGFR signaling and ligand trafficking

Intracellular transport and processing of ligands is critical to the activation of signal transduction pathways that guide development. Star is an essential gene in Drosophila that has been implicated in the trafficking of ligands for epidermal growth factor (EGF) receptor signaling. The role of cytoplasmic motors in the endocytic and secretory pathways is well known, but the specific requirement of motors in EGF receptor transport has not been investigated. We identified Star in a screen designed to recover second-site modifiers of the dominant rough eye phenotype of the Glued mutation Gl1. The Glued (Gl) locus encodes the p150 subunit of the dynactin complex, an activator of cytoplasmic dynein-driven motility. We show that alleles of Gl and dynein genetically interact with both Star and EGFR alleles. Similarly to mutations in Star, the Gl1 mutation is capable of modifying the phenotypes of the EGFR mutation Ellipse. These genetic interactions suggest a model in which Star, dynactin and dynein cooperate in the trafficking of EGF ligands. In support of this model, overexpression of the cleaved, active Spitz ligand can partially bypass defective trafficking and suppress the genetic interactions. Our direct observations of live S2 cells show that export of Spitz-GFP from the endoplasmic reticulum, as well as the trafficking of Spitz-GFP vesicles, depends on both Star and dynein.

Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila.

Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila.

Interrogating Emergent Transport Properties for Molecular Motor Ensembles: A Semi-analytical Approach

Intracellular transport is an essential function in eucaryotic cells, facilitated by motor proteins—proteins converting chemical energy into kinetic energy. It is understood that motor proteins work in teams enabling unidirectional and bidirectional transport of intracellular cargo over long distances. Disruptions of the underlying transport mechanisms, often caused by mutations that alter single motor characteristics, are known to cause neurodegenerative diseases. For example, phosphorylation of kinesin motor domain at the serine residue is implicated in Huntington’s disease, with a recent study of phosphorylated and phosphomimetic serine residues indicating lowered single motor stalling forces. In this article we report the effects of mutations of this nature on transport properties of cargo carried by multiple wild-type and mutant motors. Results indicate that mutants with altered stall forces might determine the average velocity and run-length even when they are outnumbered by wild type motors in the ensemble. It is shown that mutants gain a competitive advantage and lead to an increase in the expected run-length when the load on the cargo is in the vicinity of the mutant’s stalling force or a multiple of its stalling force. A separate contribution of this article is the development of a semi-analytic method to analyze transport of cargo by multiple motors of multiple types. The technique determines transition rates between various relative configurations of motors carrying the cargo using the transition rates between various absolute configurations. This enables a computation of biologically relevant quantities like average velocity and run-length without resorting to Monte Carlo simulations. It can also be used to introduce alterations of various single motor parameters to model a mutation and to deduce effects of such alterations on the transport of a common cargo by multiple motors. Our method is easily implementable and we provide a software package for general use.

Kinesin transport: driving kinesin in the neuron

A plethora of cytoplasmic motors contribute to the directed transport of a wide range of cellular organelles and molecules. Recent studies have advanced our understanding of cargo attachment to motor molecules and the regulation of intracellular transport.

Drosophila cytoplasmic dynein: Mutations, tools, and developmental functions

Abstract The study of Drosophila cytoplasmic dynein has leveraged the awesome power of genetics to isolate mutations in dynein subunits and allow the organism to reveal dyneins essential functions. These studies have relied on an extensive background of literature describing Drosophila development, as well as a tool box of sophisticated technologies that have allowed the targeted studies of dynein function in specific cells and tissues. In this review, we summarize many of the available Drosophila mutations and tools that are used to study dyneins function and highlight developmental functions of dynein in germ cells, embryos, and neurons.