Competition between physical search and a weak-to-strong transition rate-limits kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions.

A new method to experimentally quantify dynamics of initial protein-protein interactions.

Cells run on initiation of protein-protein interactions, which are dynamically tuned spatially and temporally to modulate cellular events. This tuning can be physical, such as attaching the protein to a cargo or protein complex, thereby altering its diffusive properties, or modulating the distance between protein pairs, or chemical, by altering the proteins' conformations (e.g.

Host-defense piscidin peptides as antibiotic adjuvants against Clostridioides difficile.

The spore-forming intestinal pathogen Clostridioides difficile causes multidrug resistant infection with a high rate of recurrence after treatment. Piscidins 1 (p1) and 3 (p3), cationic host defense peptides with micromolar cytotoxicity against C. difficile, sensitize C.

ADP release can explain spatially-dependent kinesin binding times.

The self-organization of cells relies on the profound complexity of protein-protein interactions. Challenges in directly observing these events have hindered progress toward understanding their diverse behaviors. One notable example is the interaction between molecular motors and cytoskeletal systems that combine to perform a variety of cellular functions.

The ubiquitous microtubule-associated protein 4 (MAP4) controls organelle distribution by regulating the activity of the kinesin motor.

Regulation of organelle transport by molecular motors along the cytoskeletal microtubules is central to maintaining cellular functions. Here, we show that the ubiquitous tau-related microtubule-associated protein 4 (MAP4) can bias the bidirectional transport of organelles toward the microtubule minus-ends. This is concurrent with MAP4 phosphorylation, mediated by the kinase GSK3β.

Developing Antimicrobial Synergy With AMPs.

Antimicrobial peptides (AMPs) have been extensively studied due to their vast natural abundance and ability to kill microbes. In an era critically lacking in new antibiotics, manipulating AMPs for therapeutic application is a promising option. However, bacterial pathogens resistant to AMPs remain problematic.

A novel antibacterial strategy: histone and antimicrobial peptide synergy.

The rate at which antibiotics are discovered and developed has stagnated; meanwhile, antibacterial resistance continually increases and leads to a plethora of untreatable and deadly infections worldwide. Therefore, there is a critical need to develop new antimicrobial strategies to combat this alarming reality. One approach is to understand natural antimicrobial defense mechanisms that higher-level organisms employ in order to kill bacteria, potentially leading to novel antibiotic therapeutic approaches.

Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense.

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs.

Mammalian histones facilitate antimicrobial synergy by disrupting the bacterial proton gradient and chromosome organization.

First proposed as antimicrobial agents, histones were later recognized for their role in condensing chromosomes. Histone antimicrobial activity has been reported in innate immune responses. However, how histones kill bacteria has remained elusive.

Regulation of in vivo dynein force production by CDK5 and 14-3-3ε and KIAA0528.

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.

Combined Positive and Negative Feedback Allows Modulation of Neuronal Oscillation Frequency during Sensory Processing.

A key step in sensory information processing involves modulation and integration of neuronal oscillations in disparate frequency bands, a poorly understood process. Here, we investigate how top-down input causes frequency changes in slow oscillations during sensory processing and, in turn, how the slow oscillations are combined with fast oscillations (which encode sensory input). Using experimental connectivity patterns and strengths of interneurons, we develop a system-level model of a neuronal circuit controlling these oscillatory behaviors, allowing us to understand the mechanisms responsible for the observed oscillatory behaviors.

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

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.

Cargo navigation across 3D microtubule intersections.

The eukaryotic cell's microtubule cytoskeleton is a complex 3D filament network. Microtubules cross at a wide variety of separation distances and angles. Prior studies in vivo and in vitro suggest that cargo transport is affected by intersection geometry.

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.

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.

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.

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5.

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction--including switch-1 and the neck linker--how movement of these two domains is coordinated through the ATPase cycle remains unknown.

Microtubule C-Terminal Tails Can Change Characteristics of Motor Force Production.

Control of intracellular transport is poorly understood, and functional ramifications of tubulin isoform differences between cell types are mostly unexplored. Motors' force production and detachment kinetics are critical for their group function, but how microtubule (MT) details affect these properties--if at all--is unknown. We investigated these questions using both a vesicular transport human kinesin, kinesin-1, and also a mitotic kinesin likely optimized for group function, kinesin-5, moving along either bovine brain or MCF7(breast cancer) MTs.

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.

Autoregulatory mechanism for dynactin control of processive and diffusive dynein transport.

Dynactin is the longest known cytoplasmic dynein regulator, with roles in dynein recruitment to subcellular cargo and in stimulating processive dynein movement. The latter function was thought to involve the N-terminal microtubule-binding region of the major dynactin polypeptide p150(Glued), although recent results disputed this. To understand how dynactin regulates dynein we generated recombinant fragments of the N-terminal half of p150(Glued).

Calibration of optical tweezers for in vivo force measurements: how do different approaches compare?

There is significant interest in quantifying force production inside cells, but since conditions in vivo are less well controlled than those in vitro, in vivo measurements are challenging. In particular, the in vivo environment may vary locally as far as its optical properties, and the organelles manipulated by the optical trap frequently vary in size and shape. Several methods have been proposed to overcome these difficulties.

CK2 activates kinesin via induction of a conformational change.

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain.

A biophysical analysis of mitochondrial movement: differences between transport in neuronal cell bodies versus processes.

There is an increasing interest in factors that can impede cargo transport by molecular motors inside the cell. Although potentially relevant (Yi JY, Ori-McKenney KM, McKenney RJ, Vershinin M, Gross SP, Vallee RB. High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport.

Review: biogenesis of the multifunctional lipid droplet: lipids, proteins, and sites.

Lipid droplets (LDs) are ubiquitous dynamic organelles that store and supply lipids in all eukaryotic and some prokaryotic cells for energy metabolism, membrane synthesis, and production of essential lipid-derived molecules. Interest in the organelle's cell biology has exponentially increased over the last decade due to the link between LDs and prevalent human diseases and the discovery of new and unexpected functions of LDs. As a result, there has been significant recent progress toward understanding where and how LDs are formed, and the specific lipid pathways that coordinate LD biogenesis.

Axonal transport: how high microtubule density can compensate for boundary effects in small-caliber axons.

Long-distance intracellular axonal transport is predominantly microtubule-based, and its impairment is linked to neurodegeneration. In this study, we present theoretical arguments 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. Our theoretical analysis suggests that this opposition to motion increases rapidly as the cargo approaches the wall.