Land of the Rising Chlamy: a report of the 11th international meeting on the Cell and Molecular Biology of Chlamydomonas held in Kobe, Japan, from May 11--15, 2004.
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Animals↗
Biomedical subjects
Stephen M King
Publications and source records attributed to Stephen M King.
Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein.
The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.
Adenosine Triphosphate↗
Relaxation-based structure refinement and backbone molecular dynamics of the dynein motor domain-associated light chain.
The light chain 1 (LC1) polypeptide is a member of the leucine-rich repeat protein family and binds at or near the ATP hydrolytic site within the motor domain of the gamma heavy chain from Chlamydomonas outer arm dynein. It consists of an N-terminal helix, a central barrel formed from six leucine-rich repeats that fold as beta beta alpha units, and a C-terminal helical domain that protrudes from the main axis defined by the leucine-rich repeats. Interaction with the gamma heavy chain is likely mediated through a hydrophobic patch on the larger beta sheet face, and the C-terminal region is predicted to insert into the dynein ATP hydrolytic site. Here we have used 1H-15N heteronuclear relaxation measurements obtained at 500 and 600 MHz to refine and validate the LC1 solution structure. In this refined structure, the C-terminal helix is significantly reoriented by more than 20 degrees as compared to the control and provides a more precise understanding of the potential regulatory role of this domain. We also employed the refined structure to perform a dynamic analysis of LC1 using the 600 MHz data set. These results, which were cross validated using the 500 MHz data set, strongly support identification of the predicted LC1 binding surfaces and provide additional insight into the interaction mechanisms of leucine-rich repeat proteins.
Animals↗
Backbone dynamics of dynein light chains.
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Animals↗
Organization and regulation of the dynein microtubule motor.
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Animals↗
A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72.
Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.
Amino Acid Sequence↗
The roadblock light chain binds a novel region of the cytoplasmic Dynein intermediate chain.
Cytoplasmic dynein is the major minus-end directed microtubule-based motor in eukaryotic cells. It is composed of a number of different subunits including three light chain families: Tctex1, LC8, and roadblock. The incorporation of the roadblock light chains into the cytoplasmic dynein complex had not been determined. There are two roadblock genes in mammals, ROBL-1 and ROBL-2. We find that both members of the roadblock family bind directly to all of the intermediate chain isoforms of mammalian cytoplasmic dynein. This was determined with three complementary approaches. A yeast two-hybrid assay demonstrated that both roadblock light chains interact with intermediate chain isoforms from the IC74-1 and IC74-2 genes in vivo. This was confirmed in vitro with both a solid phase blot overlay assay and a solution-binding assay. The roadblock-binding domain on the intermediate chain was mapped to an approximately 72 residue region. The binding domain is downstream of each of the two alternative splice sites in the intermediate chains. This location is consistent with the finding that both roadblock-1 and roadblock-2 show no binding specificity for a single IC74-1 or IC74-2 intermediate chain isoform. In addition, this roadblock-binding domain is significantly downstream from both the Tctex1- and LC8-binding sites, supporting the hypothesis that multiple light chain family members can bind to the same intermediate chain.
Binding Sites↗
Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.
The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.
Amino Acid Sequence↗
Dyneins motor on in plants.
A recent report based on analysis of the Arabidopsis genome suggested that angiosperms do not contain the dynein microtubule motor. However, examination of the whole genome shotgun sequence for rice (Oryza sativa) has revealed that four dynein heavy chains are present in this monocot, indicating that the apparent lack of these sequences in Arabidopsis is not a general feature of angiosperm genomic organization. These observations also suggest that, in contrast to an earlier proposal, flowering plants may indeed use standard dynein-driven mechanisms to perform cellular transport activities that, in other organisms, employ the dynein motor
Amino Acid Sequence↗
Design and regulation of the AAA+ microtubule motor dynein.
Dyneins are highly complex molecular motors that transport their attached cargo towards the minus end of microtubules. These enzymes are required for many essential motile activities within the cytoplasm and also power eukaryotic cilia and flagella. Each dynein contains one or more heavy chain motor units that consist of an N-terminal stem domain that is involved in cargo attachment, and six AAA+ domains (AAA1-6) plus a C-terminal globular segment that are arranged in a heptameric ring. At least one AAA+ domain (AAA1) is capable of ATP binding and hydrolysis, and the available data suggest that one or more additional domains also may bind nucleotide. The ATP-sensitive microtubule binding site is located at the tip of a 10nm coiled coil stalk that emanates from between AAA4 and AAA5. The function of this motor both in the cytoplasm and the flagellum must be tightly regulated in order to result in useful work. Consequently, dyneins also contain a series of additional components that serve to define the cargo-binding properties of the enzyme and which act as sensors to transmit regulatory inputs to the motor units. Here we describe the two basic dynein designs and detail the various regulatory systems that impinge on this motor within the eukaryotic flagellum.
Binding Sites↗