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.

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.

Summary Lipid droplets (LDs) are dynamic organelles that collect, store, and supply lipids [1]. LDs have a central role in the exchange of lipids occurring between the cell and the environment, and provide cells with substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules such as lipoproteins or hormones. However, lipid-derived metabolites also cause progressive lipotoxicity [2]; accumulation of reactive oxygen species (ROS), endoplasmic reticulum stress, mitochondrial malfunctioning, and cell death [2]. Intracellular accumulation of LDs is a hallmark of prevalent human diseases including obesity, steatosis, diabetes, myopathies, and arteriosclerosis [3]. Indeed, non-alcoholic fatty liver disease is the most common cause of abnormal hepatic function among adults [4, 5]. Lipotoxicity gradually promotes cellular ballooning and disarray, megamitochondria, and accumulation of Mallory’s hyaline in hepatocytes and inflammation, fibrosis, and cirrhosis in the liver. Here, using confocal microscopy, serial-block-face scanning electron microscopy, and flow-cytometry we show that LD accumulation is heterogeneous within a cell population and follows a positive skewed distribution. Lipid availability and fluctuations in biochemical networks controlling lipolysis, fatty acid oxidation, and protein synthesis, contribute to cell-to-cell heterogeneity. Critically, this reversible variability generates a subpopulation of cells that effectively collect and store lipids. This high-lipid subpopulation accumulates more LDs, more ROS, and reduces the risk of lipotoxicity to the population without impairing overall lipid homeostasis, since high-lipid cells can supply stored lipids to the other cells. In conclusion, we demonstrate fat storage compartmentalization within a cell population and propose that this is a protective social organization to reduce lipotoxicity.

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.

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.

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.

Single-molecule cytoplasmic dynein function is well understood, but there are major gaps in mechanistic understanding of cellular dynein regulation. We reported a mode of dynein regulation, force adaptation, where lipid droplets adapt to opposition to motion by increasing the duration and magnitude of force production, and found LIS1 and NudEL to be essential. Adaptation reflects increasing NudEL-LIS1 utilization; here, we hypothesize that such increasing utilization reflects CDK5-mediated NudEL phosphorylation, which increases the dynein-NudEL interaction, and makes force adaptation possible. We report that CDK5, 14-3-3ε, and CDK5 cofactor KIAA0528 together promote NudEL phosphorylation and are essential for force adaptation. By studying the process in COS-1 cells lacking Tau, we avoid confounding neuronal effects of CDK5 on microtubules. Finally, we extend this in vivo regulatory pathway to lysosomes and mitochondria. Ultimately, we show that dynein force adaptation can control the severity of lysosomal tug-of-wars among other intracellular transport functions involving high force.

Cellular cargos, including lipid droplets and mitochondria, are transported along microtubules using molecular motors such as kinesins. In the cell, it is unclear how motors are coordinated to achieve transport outcomes that are cargo-specific. One possibility is that transport is modulated by differences in organization and mobility of motors on the cargo's surface. We use mechanochemical 3D computational modeling to compare different motor anchoring modes, and find that organizational changes can optimize for different objectives. Cargos with clustered motors are transported efficiently, but are slow to bind to microtubules. Cargos with motors dispersed rigidly on their surface bind microtubules quickly, but are transported inefficiently. Cargos with freely-diffusing motors have both fast binding and efficient transport, although less efficient than clustered motors. These results point to a functional role for observed changes in motor organization on cargos, and suggest motor diffusivity as a control point for transport, either by modulation of adaptor proteins or changes in lipid composition. Author summaryThe molecular motors of the kinesin family are responsible for moving the parts of cells 1 to their subcellular destinations, organizing the cell interior. Computational modeling 2 has historically played an important role in understanding how these motors work. But, 3 strikingly, most models assume the subcellular cargos have rigid surfaces. Simulating 4 fluid surfaces is more challenging but more applicable to biological cargo. In this work, 5 we build a computational model of molecular motor transport of cargo with fluid 6 surfaces. Surface fluidity gives cargo transport new properties, like enhanced transport 7 efficiency, and the ability to push against opposing forces. Because the effects of surface 8 fluidity vary strongly with some of the properties of the cargo, like its size, our results 9 hint at one of the ways that the same transport machinery can distinguish different 10 cargo despite using the same motors. 13 kinesin and dynein superfamilies to transport organelles and other cargo along 14 microtubules. Despite having only a limited set of cargo transport motors (kinesin-1, 15 kinesin-2 and kinesin-3 families [1], along with cytoplasmic dynein), different cargos are 16 transported to different locations, even though they are transported along the same set 17 of microtubule "roads". For example, under normal conditions COS-7 cells direct lipid 18 droplets toward microtubule plus end, localizing them near the plasma membrane, and 19 mitochondria toward the minus end, localizing them near the nucleus. Under glucose 20 starvation, localization of both organelles changes to spread them out around the cell, 21 allowing them to come into contact with each other [2]. How do cells achieve these 22 cargo-specific routing outcomes? In some cases, cells use molecular specificity to achieve 23 cargo specificity, such as using specific linkers or cargo-bound regulators [3,4]. 24 However, recent experiment...