Babu J.N. Reddy

Acyl-CoA synthetase 3 promotes lipid droplet biogenesis in ER microdomains

Acyl-CoA synthetase 3 is recruited early to lipid droplet assembly sites on the ER, where it is required for efficient lipid droplet nucleation and lipid storage.

AMPK activation promotes lipid droplet dispersion on detyrosinated microtubules to increase mitochondrial fatty acid oxidation

Lipid droplets (LDs) are intracellular organelles that provide fatty acids (FAs) to cellular processes including synthesis of membranes and production of metabolic energy. While known to move bidirectionally along microtubules (MTs), the role of LD motion and whether it facilitates interaction with other organelles are unclear. Here we show that during nutrient starvation, LDs and mitochondria relocate on detyrosinated MT from the cell centre to adopt a dispersed distribution. In the cell periphery, LD–mitochondria interactions increase and LDs efficiently supply FAs for mitochondrial beta-oxidation. This cellular adaptation requires the activation of the energy sensor AMPK, which in response to starvation simultaneously increases LD motion, reorganizes the network of detyrosinated MTs and activates mitochondria. In conclusion, we describe the existence of a specialized cellular network connecting the cellular energetic status and MT dynamics to coordinate the functioning of LDs and mitochondria during nutrient scarcity.

Load-induced enhancement of Dynein force production by LIS1–NudE in vivo and in vitro

Most sub-cellular cargos are transported along microtubules by kinesin and dynein molecular motors, but how transport is regulated is not well understood. It is unknown whether local control is possible, for example, by changes in specific cargo-associated motor behaviour to react to impediments. Here we discover that microtubule-associated lipid droplets (LDs) in COS1 cells respond to an optical trap with a remarkable enhancement in sustained force production. This effect is observed only for microtubule minus-end-moving LDs. It is specifically blocked by RNAi for the cytoplasmic dynein regulators LIS1 and NudE/L (Nde1/Ndel1), but not for the dynactin p150Glued subunit. It can be completely replicated using cell-free preparations of purified LDs, where duration of LD force production is more than doubled. These results identify a novel, intrinsic, cargo-associated mechanism for dynein-mediated force adaptation, which should markedly improve the ability of motor-driven cargoes to overcome subcellular obstacles.

Casein kinase 2 reverses tail-independent inactivation of kinesin-1

Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signaling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (~ 4-fold) without altering kinesin’s single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase up-regulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.

A posttranslational modification of the mitotic kinesin Eg5 that enhances its mechanochemical coupling and alters its mitotic function

Significance Members of the kinesin superfamily serve a wide variety of functions, and a dominant narrative for these molecular motors has been that each member of the superfamily is uniquely specialized to serve a very limited set of functions. However, it is now appreciated that many members of this group serve several distinct physiological roles, and it has been unclear how these kinesins accomplish this functional flexibility. In this report, we describe a posttranslational modification of the kinesin 5 family member Eg5 that dramatically alters its chemomechanical behavior to make it function much more efficiently under load and in ensembles. This work provides the biophysical context required to mechanistically understand the effects of modified Eg5 in dividing cells. Numerous posttranslational modifications have been described in kinesins, but their consequences on motor mechanics are largely unknown. We investigated one of these—acetylation of lysine 146 in Eg5—by creating an acetylation mimetic lysine to glutamine substitution (K146Q). Lysine 146 is located in the α2 helix of the motor domain, where it makes an ionic bond with aspartate 91 on the neighboring α1 helix. Molecular dynamics simulations predict that disrupting this bond enhances catalytic site–neck linker coupling. We tested this using structural kinetics and single-molecule mechanics and found that the K146Q mutation increases motor performance under load and coupling of the neck linker to catalytic site. These changes convert Eg5 from a motor that dissociates from the microtubule at low load into one that is more tightly coupled and dissociation resistant—features shared by kinesin 1. These features combined with the increased propensity to stall predict that the K146Q Eg5 acetylation mimetic should act in the cell as a “brake” that slows spindle pole separation, and we have confirmed this by expressing this modified motor in mitotically active cells. Thus, our results illustrate how a posttranslational modification of a kinesin can be used to fine tune motor behavior to meet specific physiological needs.

Heterogeneity in kinesin function

The kinesin family proteins are often studied as prototypical molecular motors; a deeper understanding of them can illuminate regulation of intracellular transport. It is typically assumed that they function identically. Here we find that this assumption of homogeneous function appears incorrect: variation among motors’ velocities in vivo and in vitro is larger than the stochastic variation expected for an ensemble of “identical” motors. When moving on microtubules, slow and fast motors are persistently slow, and fast, respectively. We develop theory that provides quantitative criteria to determine whether the observed single‐molecule variation is too large to be generated from an ensemble of identical molecules. To analyze such heterogeneity, we group traces into homogeneous sub‐ensembles. Motility studies varying the temperature, pH and glycerol concentration suggest at least 2 distinct functional states that are independently affected by external conditions. We end by investigating the functional ramifications of such heterogeneity through Monte‐Carlo multi‐motor simulations.

Axonal Transport: A Constrained System

Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. Here we review recent theoretical and experimental evidence that suggest that near the axon boundaries (walls), the effective viscosity can become large enough to impede cargo transport in small (but not large) caliber axons. Theoretical work suggests that this opposition to motion increases rapidly as the cargo approaches the wall. However, having parallel microtubules close enough together to enable a cargo to simultaneously engage motors on more than one microtubule dramatically enhances motor activity, and thus decreases the effects due to such opposition. Experimental evidence supports this hypothesis: in small caliber axons, microtubule density is higher, increasing the probability of having parallel microtubules close enough that they can be used simultaneously by motors on a cargo. For transport toward the minus-end of microtubules, e.g., toward the cell body in an axon, a recently discovered force adaptation system can also contribute to overcoming such opposition to motion.

Dynein: Let's not get stuck!

To take advantage of the different chemical environments made possible by internal membrane bound sub-compartments, Eukaryotic cells heavily utilize active transport. Much of this involves moving distinct cargos along microtubules, perhaps to facilitate cargo-cargo interactions—e.g. to control transfer of lipids from lipid droplets to mitochondria as part of metabolism—or to allow function of extended cells such as neurons. There are 2 major families of microtubule molecular motors: the predominantly plus-end directed kinesin motors, and minus-end directed dynein, and evolutionarily they appear to have taken a different approach to modulating function. There are many kinesin-family members, and these appear to have been adapted to specific cellular functions. In contrast, there are typically only one or at most 2 genes for cytoplasmic dynein, and the multiple distinct functions of dynein are altered by functioning with the 2 large cofactor complexes, Dynactin and NudE/Lis1. Dynactin was originally discovered to increase dynein travel distances, and was later reported to also have a domain able to turn off dynein force production, suggesting multiple levels of potential regulation that have yet to be explored. The NudE/Lis1 system was identified to play an important role in development and disease, as mutations in the Lis1 protein result in a devastating developmental defect known as Miller-Dieker Syndrome, resulting from impaired neuronal migration due to impaired nuclear migration. Later studies in vitro showed that together with NudE, Lis1 modulates dynein force production, allowing the motor to better remain attached to microtubules when under load. By decreasing the motors’ detachment rate when exposed to opposing forces, this in turn allows multiple motors to better add their forces, resulting in improved ensemble function, and thus better ability to move cargos against opposition. While this in vitro study appeared to potentially explain the in vivo Miller-Dieker lissencephaly phenotype—neuronal migration requires transport of the cells’ nucleus down the axon, a dynein-dependent process likely facing serious opposition, and thus such migration is likely dependent on improved dynein function—a number of key questions remained. First, it was unknown whether cargo transport was relatively constant, and insensitive to local environmental factors, or whether there were in fact some local sensing and response, e.g., to impediments to motion. Second, it remained to be determined whether the NudE/Lis1 system altered dynein the same way in vivo as it did in vitro, given the more complicated in vivo environment. Third, the extent to which the NudE/Lis1 system was important for cargos other than nuclei remained an open question. Finally, whether—and how—the system worked in conjunction with the dynactin complex was unknown.