Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein.
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ABSTRACT:
SUBMITTER: Toba S
PROVIDER: S-EPMC1424663 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytoplasmic dynein is the most complex cytoskeletal motor protein and is responsible for numerous biological functions. Essential to dynein's function is its capacity to respond anisotropically to tension, so that its microtubule-binding domains bind microtubules more strongly when under backward load than forward load. The structural mechanisms by which dynein senses directional tension, however, are unknown. Using a combination of optical tweezers, mutagenesis, and chemical cross-linking, we show that three structural elements protruding from the motor domain-the linker, buttress, and stalk-together regulate directional tension-sensing. We demonstrate that dynein's anisotropic response to directional tension is mediated by sliding of the coiled-coils of the stalk, and that coordinated conformational changes of dynein's linker and buttress control this process. We also demonstrate that the stalk coiled-coils assume a previously undescribed registry during dynein's stepping cycle. We propose a revised model of dynein's mechanochemical cycle which accounts for our findings.
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Project description:The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis-Menten equation. In the absence of products, the K(M) (the [ATP] required for half-maximal speed) was 28 +/- 1 microM, and the maximum speed was 904 nm/s. P(i) behaved as a competitive inhibitor with K(I) = 9 +/- 1 mM. ADP behaved approximately as a competitive inhibitor with K(I) = 35 +/- 2 microM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P(i) is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P(i) before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P(i) release from the other.
| S-EPMC337027 | biostudies-literature
Project description:Cytoplasmic dynein is a molecular motor responsible for minus-end-directed cargo transport along microtubules (MTs). Dynein motility has previously been studied on surface-immobilized MTs in vitro, which constrains the motors to move in two dimensions. In this study, we explored dynein motility in three dimensions using an MT bridge assay. We found that dynein moves in a helical trajectory around the MT, demonstrating that it generates torque during cargo transport. Unlike other cytoskeletal motors that produce torque in a specific direction, dynein generates torque in either direction, resulting in bidirectional helical motility. Dynein has a net preference to move along a right-handed helical path, suggesting that the heads tend to bind to the closest tubulin binding site in the forward direction when taking sideways steps. This bidirectional helical motility may allow dynein to avoid roadblocks in dense cytoplasmic environments during cargo transport.
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Project description:Until recently, dynein was the least understood of the cytoskeletal motors. However, a wealth of new structural, mechanistic, and cell biological data is shedding light on how this complicated minus-end-directed, microtubule-based motor works. Cytoplasmic dynein-1 performs a wide array of functions in most eukaryotes, both in interphase, in which it transports organelles, proteins, mRNAs, and viruses, and in mitosis and meiosis. Mutations in dynein or its regulators are linked to neurodevelopmental and neurodegenerative diseases. Here, we begin by providing a synthesis of recent data to describe the current model of dynein's mechanochemical cycle. Next, we discuss regulators of dynein, with particular focus on those that directly interact with the motor to modulate its recruitment to microtubules, initiate cargo transport, or activate minus-end-directed motility.
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Project description:Cytoplasmic dynein is a molecular motor that transports a large variety of cargoes (e.g., organelles, messenger RNAs, and viruses) along microtubules over long intracellular distances. The dynactin protein complex is important for dynein activity in vivo, but its precise role has been unclear. Here, we found that purified mammalian dynein did not move processively on microtubules in vitro. However, when dynein formed a complex with dynactin and one of four different cargo-specific adapter proteins, the motor became ultraprocessive, moving for distances similar to those of native cargoes in living cells. Thus, we propose that dynein is largely inactive in the cytoplasm and that a variety of adapter proteins activate processive motility by linking dynactin to dynein only when the motor is bound to its proper cargo.
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Project description:In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome by attaching a promiscuous biotin ligase ('BioID') to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires 'activators', of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos.
| S-EPMC5533585 | biostudies-literature
Project description:Cytoplasmic dynein is the major minus end-directed microtubule motor in animal cells, and associates with many of its cargoes in conjunction with the dynactin complex. Interaction between cytoplasmic dynein and dynactin is mediated by the binding of cytoplasmic dynein intermediate chains (CD-IC) to the dynactin subunit, p150(Glued). We have found that both CD-IC and p150(Glued) are cleaved by caspases during apoptosis in cultured mammalian cells and in Xenopus egg extracts. Xenopus CD-IC is rapidly cleaved at a conserved aspartic acid residue adjacent to its NH(2)-terminal p150(Glued) binding domain, resulting in loss of the otherwise intact cytoplasmic dynein complex from membranes. Cleavage of CD-IC and p150(Glued) in apoptotic Xenopus egg extracts causes the cessation of cytoplasmic dynein--driven endoplasmic reticulum movement. Motility of apoptotic membranes is restored by recruitment of intact cytoplasmic dynein and dynactin from control cytosol, or from apoptotic cytosol supplemented with purified cytoplasmic dynein--dynactin, demonstrating the dynamic nature of the association of cytoplasmic dynein and dynactin with their membrane cargo.
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Project description:Glycogen synthase kinase 3 (GSK-3) has been linked to regulation of kinesin-dependent axonal transport in squid and flies, and to indirect regulation of cytoplasmic dynein. We have now found evidence for direct regulation of dynein by mammalian GSK-3β in both neurons and non-neuronal cells. GSK-3β coprecipitates with and phosphorylates mammalian dynein. Phosphorylation of dynein intermediate chain (IC) reduces its interaction with Ndel1, a protein that contributes to dynein force generation. Two conserved residues, S87/T88 in IC-1B and S88/T89 in IC-2C, have been identified as GSK-3 targets by both mass spectrometry and site-directed mutagenesis. These sites are within an Ndel1-binding domain, and mutation of both sites alters the interaction of IC's with Ndel1. Dynein motility is stimulated by (i) pharmacological and genetic inhibition of GSK-3β, (ii) an insulin-sensitizing agent (rosiglitazone) and (iii) manipulating an insulin response pathway that leads to GSK-3β inactivation. Thus, our study connects a well-characterized insulin-signaling pathway directly to dynein stimulation via GSK-3 inhibition.
| S-EPMC4543430 | biostudies-literature
Project description:Molecular motors are responsible for active transport and organization in the cell, underlying an enormous number of crucial biological processes. Dynein is more complicated in its structure and function than other motors. Recent experiments have found that, unlike other motors, dynein can take different size steps along microtubules depending on load and ATP concentration. We use Monte Carlo simulations to model the molecular motor function of cytoplasmic dynein at the single-molecule level. The theory relates dynein's enzymatic properties to its mechanical force production. Our simulations reproduce the main features of recent single-molecule experiments that found a discrete distribution of dynein step sizes, depending on load and ATP concentration. The model reproduces the large steps found experimentally under high ATP and no load by assuming that the ATP binding affinities at the secondary sites decrease as the number of ATP bound to these sites increases. Additionally, to capture the essential features of the step-size distribution at very low ATP concentration and no load, the ATP hydrolysis of the primary site must be dramatically reduced when none of the secondary sites have ATP bound to them. We make testable predictions that should guide future experiments related to dynein function.
| S-EPMC1189307 | biostudies-literature
Project description:We present here a simple theoretical model for conventional kinesin. The model reproduces the hand-over-hand mechanism for kinesin walking to the plus end of a microtubule. A large hindering force induces kinesin to walk slowly to the minus end, again by a hand-over-hand mechanism. Good agreement is obtained between the calculated and experimental results on the external force dependence of the walking speed, the forward/backward step ratio, and dwell times for both forward and backward steps. The model predicts that both forward and backward motions of kinesin take place at the same chemical state of the motor heads, with the front head being occupied by an ATP (or ADP,Pi) and the rear being occupied by an ADP. The direction of motion is a result of the competition between the power stroke produced by the front head and the external load. The other predictions include the external force dependence of the chemomechanical coupling ratio (e.g., the stepping distance/ATP ratio) and the walking speed of kinesin at force ranges that have not been tested by experiments. The model predicts that the chemomechanical coupling remains tight in a large force range. However, when the external force is very large (e.g., approximately 18 pN), kinesin slides in an inchworm fashion, and the translocation of kinesin becomes loosely coupled to ATP turnovers.
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