Biomedical subjects

Trina A Schroer

Publications and source records attributed to Trina A Schroer.

13 recordsLinked to original sources

PubMed2006

Dynactin enhances the processivity of kinesin-2.

Kinesin-2 is a major microtubule-based motor in most cell types. Its in vitro motile properties have been analyzed extensively and been found to differ considerably from kinesin-1. Although recombinant kinesin-2 heterodimers exhibit processive movement, the processivity of the native kinesin-2 holoenzyme has never been evaluated. Kinesin-2 can interact with dynactin, a 'processivity factor' for cytoplasmic dynein, which may alter its motile properties. In this study, we analyze the in vitro motility of single native kinesin-2 molecules and determine the effects of dynactin on motor processivity. We find that individual native kinesin-2 molecules travel processively. Dynactin has no effect on velocity but significantly increases the run length of kinesin-2 movements. These results show that the interaction with dynactin has important functional consequences on the activity of the kinesin-2 motor.

Animals↗

PubMed2006

Two-dimensional averaged images of the dynactin complex revealed by single particle analysis.

The dynactin complex interacts with dynein and numerous other proteins to provide for a wide range of subcellular transport functions. A detailed understanding of the structure and subunit organization of dynactin should yield new insights into its function. In the present study, we used single particle analysis to obtain a two-dimensional averaged image of dynactin isolated from chick embryo brains and visualized by negative stain electron microscopy (EM). Each individual image, consisting of the shoulder/sidearm and the rod, closely resembled the previously published quick-freeze deep-etch rotary-shadow electron micrographs. However, the averaged image revealed novel structural features that may have functional significance. The bulky shoulder complex has a triangular shape and is 13 nm wide and 8 nm high. The rod, with an overall length of 40 nm, consists of clearly defined lobes that are apparently grouped into three parts, the pointed-end complex, the middle segment, and the extra lobes at the barbed end. The pointed-end complex reveals the characteristic protrusions and clefts that were previously observed only in the isolated pointed-end complex. In the middle segment, the seven lobes are fitted to the helical symmetry of F-actin. A narrow but prominent gap separates the previously unidentified extra three lobes at the barbed end from the middle segment. The averaged image we obtained contrasts dramatically with the simple Arp1 polymer that was previously reported by single particle analysis of bovine brain dynactin. These apparent structural differences are probably due to the greater stability and integrity of the chick embryo brain dynactin preparation. We propose a new structural model for dynactin, based on our observations.

Actin Cytoskeleton↗

PubMed2005

Cytoplasmic dynein nomenclature.

A variety of names has been used in the literature for the subunits of cytoplasmic dynein complexes. Thus, there is a strong need for a more definitive consensus statement on nomenclature. This is especially important for mammalian cytoplasmic dyneins, many subunits of which are encoded by multiple genes. We propose names for the mammalian cytoplasmic dynein subunit genes and proteins that reflect the phylogenetic relationships of the genes and the published studies clarifying the functions of the polypeptides. This nomenclature recognizes the two distinct cytoplasmic dynein complexes and has the flexibility to accommodate the discovery of new subunits and isoforms.

Animals↗

PubMed2005

Kinesin-2 is a motor for late endosomes and lysosomes.

The bidirectional nature of late endosome/lysosome movement suggests involvement of at least two distinct motors, one minus-end directed and one plus-end directed. Previous work has identified dynein as the minus-end-directed motor for late endosome/lysosome localization and dynamics. Conventional kinesin (kinesin-1) has been implicated in plus-end-directed late endosome/lysosome movement, but other kinesin family members may also be involved. Kinesin-2 is known to drive the movement of pigment granules, a type of lysosomally derived organelle, and was recently found to be associated with purified late endosomes. To determine whether kinesin-2 might also power endosome movement in non-pigmented cells, we overexpressed dominant negative forms of the KIF3A motor subunit and KAP3 accessory subunit and knocked down KAP3 levels using RNAi. We found kinesin-2 to be required for the normal steady-state localization of late endosomes/lysosomes but not early endosomes or recycling endosomes. Despite the abnormal subcellular distribution of late endosomes/lysosomes, the uptake and trafficking of molecules through the conventional endocytic pathway appeared to be unaffected. The slow time-course of inhibition suggests that both kinesin-2 itself and its attachment to membranes do not turn over quickly.

Animals↗

PubMed2004

Fractionation and characterization of kinesin II species in vertebrate brain.

Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.

Adenosine Triphosphate↗

PubMed2004

Dynactin.

Dynactin is a multisubunit protein complex that is required for most, if not all, types of cytoplasmic dynein activity in eukaryotes. Dynactin binds dynein directly and allows the motor to traverse the microtubule lattice over long distances. A single dynactin subunit, p150Glued, is sufficient for both activities, yet dynactin contains several other subunits that are organized into an elaborate structure. It is currently believed that the bulk of the dynactin structure participates in interactions with a wide range of cellular structures, many of which are cargoes of the dynein motor. Genetic studies verify the importance of all elements of dynactin structure to its function. Although dynein can bind some membranous cargoes independently of dynactin, establishment of a fully functional dynein-cargo link appears to depend on dynactin. In this review, I summarize what is presently known about dynactin structure, the cellular structures with which it associates, and the intermolecular interactions that underlie and regulate binding. Although the molecular details of dynactin's interactions with membranous organelles and other molecules are complex, the framework provided here is intended to distill what is presently known and to be of use to dynactin specialists and beginners alike.

Animals↗

PubMed2003

Analysis of the dynein-dynactin interaction in vitro and in vivo.

Cytoplasmic dynein and dynactin are megadalton-sized multisubunit molecules that function together as a cytoskeletal motor. In the present study, we explore the mechanism of dynein-dynactin binding in vitro and then extend our findings to an in vivo context. Solution binding assays were used to define binding domains in the dynein intermediate chain (IC) and dynactin p150Glued subunit. Transient overexpression of a series of fragments of the dynein IC was used to determine the importance of this subunit for dynein function in mammalian tissue culture cells. Our results suggest that a functional dynein-dynactin interaction is required for proper microtubule organization and for the transport and localization of centrosomal components and endomembrane compartments. The dynein IC fragments have different effects on endomembrane localization, suggesting that different endomembranes may bind dynein via distinct mechanisms.

Animals↗

PubMed2003

Interactions between the evolutionarily conserved, actin-related protein, Arp11, actin, and Arp1.

The dynein activator dynactin is a multiprotein complex with distinct microtubule- and cargo-binding domains. The cargo-binding domain contains a short, actin-like filament of the actin-related protein Arp1, a second actin-related protein, Arp11, and conventional actin. The length of this filament is invariant in dynactin isolated from multiple species and tissues, suggesting that activities that regulate Arp1 polymerization are important for dynactin assembly. Arp11 is present in a protein complex localized at the pointed end of the Arp1 minifilament, whereas actin capping protein (CapZ) is present at the barbed end. Either might cooperate with conventional actin to cap Arp1. We tested the ability of Arp11 to interact with conventional actin and found it could coassemble. Like Arp1, cytosolic Arp11 is found only in dynactin, suggesting that Arp11 and free cytosolic actin do not interact significantly. Recombinant Arp11 and Arp1 were demonstrated to interact by coprecipitation. We developed an in vivo assay for Arp11-Arp1 interaction based on previous observations that Arp1 forms filamentous assemblies when overexpressed in cultured cells. Arp11 significantly decreases the formation of these organized Arp1 assemblies. Finally, this assay was used to confirm the identity of a putative Arp11 homolog in Drosophila melanogaster.

Actin-Related Protein 3↗

PubMed2003

CLIP-170 interacts with dynactin complex and the APC-binding protein EB1 by different mechanisms.

CLIP-170 is a "cytoplasmic linker protein" implicated in endosome-microtubule interactions and in control of microtubule dynamics. CLIP-170 localizes dynamically to growing microtubule plus ends, colocalizing with the dynein activator dynactin and the APC-binding protein EB1. This shared "plus-end tracking" behavior suggests that CLIP-170 might interact with dynactin and/or EB1. We have used site-specific mutagenesis of CLIP-170 and a transfection/colocalization assay to address this question in mammalian tissue culture cells. Our results indicate that CLIP-170 interacts, directly or indirectly, with both dynactin and EB1. We find that the CLIP-170/dynactin interaction is mediated by the second metal binding motif of the CLIP-170 tail. In contrast, the CLIP-170/EB1 interaction requires neither metal binding motif. In addition, our experiments suggest that the CLIP-170/dynactin interaction occurs via the shoulder/sidearm subcomplex of dynactin and can occur in the cytosol (i.e., it does not require microtubule binding). These results have implications for the targeting of both dynactin and EB1 to microtubule plus ends. Our data suggest that the CLIP-170/dynactin interaction can target dynactin complex to microtubule plus ends, although dynactin likely also targets MT plus ends directly via the microtubule binding motif of the p150(Glued) subunit. We find that CLIP-170 mutants alter p150(Glued) localization without affecting EB1, indicating that EB1 can target microtubule plus ends independently of dynactin.

Amino Acid Motifs↗

PubMed2003

Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along microtubules: role of Rab7 and RILP.

Nascent phagosomes must undergo a series of fusion and fission reactions to acquire the microbicidal properties required for the innate immune response. Here we demonstrate that this maturation process involves the GTPase Rab7. Rab7 recruitment to phagosomes was found to precede and to be essential for their fusion with late endosomes and/or lysosomes. Active Rab7 on the phagosomal membrane associates with the effector protein RILP (Rab7-interacting lysosomal protein), which in turn bridges phagosomes with dynein-dynactin, a microtubule-associated motor complex. The motors not only displace phagosomes in the centripetal direction but, strikingly, promote the extension of phagosomal tubules toward late endocytic compartments. Fusion of tubules with these organelles was documented by fluorescence and electron microscopy. Tubule extension and fusion with late endosomes and/or lysosomes were prevented by expression of a truncated form of RILP lacking the dynein-dynactin-recruiting domain. We conclude that full maturation of phagosomes requires the retrograde emission of tubular extensions, which are generated by activation of Rab7, recruitment of RILP, and consequent association of phagosomes with microtubule-associated motors.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Distinct cell cycle-dependent roles for dynactin and dynein at centrosomes.

Centrosomal dynactin is required for normal microtubule anchoring and/or focusing independently of dynein. Dynactin is present at centrosomes throughout interphase, but dynein accumulates only during S and G2 phases. Blocking dynein-based motility prevents recruitment of dynactin and dynein to centrosomes and destabilizes both centrosomes and the microtubule array, interfering with cell cycle progression during mitosis. Destabilization of the centrosomal pool of dynactin does not inhibit dynein-based motility or dynein recruitment to centrosomes, but instead causes abnormal G1 centriole separation and delayed entry into S phase. The correct balance of centrosome-associated dynactin subunits is apparently important for satisfaction of the cell cycle mechanism that monitors centrosome integrity before centrosome duplication and ultimately governs the G1 to S transition. Our results suggest that, in addition to functioning as a microtubule anchor, dynactin contributes to the recruitment of important cell cycle regulators to centrosomes.

Animals↗

PubMed2002

Cytoplasmic dynein as a facilitator of nuclear envelope breakdown.

During prophase in higher cells, centrosomes localize to deep invaginations in the nuclear envelope in a microtubule-dependent process. Loss of nuclear membranes in prometaphase commences in regions of the nuclear envelope that lie outside of these invaginations. Dynein and dynactin complex components concentrate on the nuclear envelope prior to any changes in nuclear envelope organization. These observations suggest a model in which dynein facilitates nuclear envelope breakdown by pulling nuclear membranes and associated proteins poleward along astral microtubules leading to nuclear membrane detachment. Support for this model is provided by the finding that interference with dynein function drastically alters nuclear membrane dynamics in prophase and prometaphase.

Animals↗

PubMed2002

Subunit organization in cytoplasmic dynein subcomplexes.

Because cytoplasmic dynein plays numerous critical roles in eukaryotic cells, determining the subunit composition and the organization and functions of the subunits within dynein are important goals. This has been difficult partly because of accessory polypeptide heterogeneity of dynein populations. The motor domain containing heavy chains of cytoplasmic dynein are associated with multiple intermediate, light intermediate, and light chain accessory polypeptides. We examined the organization of these subunits within cytoplasmic dynein by separating the molecule into two distinct subcomplexes. These subcomplexes were competent to reassemble into a molecule with dynein-like properties. One subcomplex was composed of the dynein heavy and light intermediate chains whereas the other subcomplex was composed of the intermediate and light chains. The intermediate and light chain subcomplex could be further separated into two pools, only one of which contained dynein light chains. The two pools had distinct intermediate chain compositions, suggesting that intermediate chain isoforms have different light chain-binding properties. When the two intermediate chain pools were characterized by analytical velocity sedimentation, at least four molecular components were seen: intermediate chain monomers, intermediate chain dimers, intermediate chain monomers with bound light chains, and a mixture of intermediate chain dimers with assorted bound light chains. These data provide new insights into the compositional heterogeneity and assembly of the cytoplasmic dynein complex and suggest that individual dynein molecules have distinct molecular compositions in vivo.

Animals↗