Roop Mallik

Cytoplasmic dynein functions as a gear in response to load

Cytoskeletal molecular motors belonging to the kinesin and dynein families transport cargos (for example, messenger RNA, endosomes, virus) on polymerized linear structures called microtubules in the cell. These ‘nanomachines’ use energy obtained from ATP hydrolysis to generate force, and move in a step-like manner on microtubules. Dynein has a complex and fundamentally different structure from other motor families. Thus, understanding dyneins force generation can yield new insight into the architecture and function of nanomachines. Here, we use an optical trap to quantify motion of polystyrene beads driven along microtubules by single cytoplasmic dynein motors. Under no load, dynein moves predominantly with a mixture of 24-nm and 32-nm steps. When moving against load applied by an optical trap, dynein can decrease step size to 8 nm and produce force up to 1.1 pN. This correlation between step size and force production is consistent with a molecular gear mechanism. The ability to take smaller but more powerful strokes under load—that is, to shift gears—depends on the availability of ATP. We propose a model whereby the gear is downshifted through load-induced binding of ATP at secondary sites in the dynein head.

Molecular Motors: Strategies to Get Along

The majority of active transport in the cell is driven by three classes of molecular motors: the kinesin and dynein families that move toward the plus-end and minus-end of microtubules, respectively, and the unconventional myosin motors that move along actin filaments. Each class of motor has different properties, but in the cell they often function together. In this review we summarize what is known about their single-molecule properties and the possibilities for regulation of such properties. In view of new results on cytoplasmic dynein, we attempt to rationalize how these different classes of motors might work together as part of the intracellular transport machinery. We propose that kinesin and myosin are robust and highly efficient transporters, but with somewhat limited room for regulation of function. Because cytoplasmic dynein is less efficient and robust, to achieve function comparable to the other motors it requires a number of accessory proteins as well as multiple dyneins functioning together. This necessity for additional factors, as well as dyneins inherent complexity, in principle allows for greatly increased control of function by taking the factors away either singly or in combination. Thus, dyneins contribution relative to the other motors can be dynamically tuned, allowing the motors to function together differently in a variety of situations.

Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes

Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays, this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities.

Building Complexity: An In Vitro Study of Cytoplasmic Dynein with In Vivo Implications

BACKGROUND Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. RESULTS We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dyneins mechanochemical cycle. CONCLUSIONS Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.

Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes.

Large low temperature magnetoresistance and magnetic anomalies in Tb2PdSi3 and Dy2PdSi3

The results of heat-capacity ( C ), magnetic susceptibility (χ), electrical resistivity ( ρ ) and magnetoresistance (Δ ρ / ρ ) measurements on the compounds, Tb 2 PdSi 3 and Dy 2 PdSi 3 , are reported. The results establish that these compounds undergo long-range magnetic ordering (presumably with a complex magnetic structure) below ( T c =) 23 and 8 K, respectively. The Δ ρ / ρ is negative in the vicinity of T c and the magnitude grows as T c is approached from higher temperatures as in the case of well-known giant magnetoresistance systems (La manganite-based perovskites); this is attributed to the formation of some kind of magnetic polarons. The magnitude of Δ ρ / ρ at low temperatures is quite large, for instance, about 30% in the presence of 60 kOe field at 5 K in Dy sample.

Teamwork in microtubule motors.

Diverse cellular processes are driven by the collective force from multiple motor proteins. Disease-causing mutations cause aberrant function of motors, but the impact is observed at a cellular level and beyond, therefore necessitating an understanding of cell mechanics at the level of motor molecules. One way to do this is by measuring the force generated by ensembles of motors in vivo at single-motor resolution. This has been possible for microtubule motor teams that transport intracellular organelles, revealing unexpected differences between collective and single-molecule function. Here we review how the biophysical properties of single motors, and differences therein, may translate into collective motor function during organelle transport and perhaps in other processes outside transport.

Observation of a minimum in the temperature-dependent electrical resistance above the magnetic-ordering temperature in Gd2PdSi3

Results on electrical resistivity, magnetoresistance, magnetic susceptibility, heat capacity and 155Gd Mossbauer measurements on a Gd-based intermetallic compound, Gd2PdSi3, are reported. A finding of interest is that the resistivity unexpectedly shows a well-defined minimum at about 45 K, well above the long-range magnetic-ordering temperature (21 K), a feature which gets suppressed by the application of a magnetic field. This observation in a Gd alloy presents an interesting scenario. On the basis of our results, we propose electron localization induced by s-f (or d-f) exchange interaction prior to long-range magnetic order as a mechanism for the electrical resistance minimum.

LARGE POSITIVE MAGNETORESISTANCE AT LOW TEMPERATURES IN A FERROMAGNETIC NATURAL MULTILAYER, LAMN2GE2

The results of magnetoresistance measurements on a naturally occurring multilayer LaMn 2 Ge 2 , which is ferromagnetic below 326 K, are reported. The magnitude of magnetoresistance is found to be positive below 70 K gradually increasing to an unusually large value (nearly 100%) at 4.2 K in the presence of a field of 70 kOe as the temperature is lowered, similar to the recent observations by Verbanck, Temst, Mae, Schad, Van Bael, Moshchalkov, and Bruynseraede [Appl. Phys. Lett. 70, 1477 (1997)] in Cr/Ag/Cr trilayers. The positive sign of magnetoresistance for a ferromagnet is unexpected and possible explanations are offered.

Quantitative optical trapping on single organelles in cell extract.

We have developed an optical trapping method to precisely measure the force generated by motor proteins on single organelles of unknown size in cell extract. This approach, termed VMatch, permits the functional interrogation of native motor complexes. We apply VMatch to measure the force, number and activity of kinesin-1 on motile lipid droplets isolated from the liver of normally fed and food-deprived rats.