Neural Tracing Protein-Functionalized Nanoparticles Capable of Fast Retrograde Axonal Transport in Live Neurons.

Neural tracing proteins like horseradish peroxidase-conjugated wheat germ agglutinin (WGA-HRP) can target the central nervous system (CNS) through anatomic retrograde transport without crossing the blood-brain barrier (BBB). Conjugating WGA-HRP to nanoparticles may enable the creation of BBB-bypassing nanomedicine. Microfluidics and two-photon confocal microscopy is applied to screen nanocarriers for transport efficacy and gain mechanistic insights into their interactions with neurons. Protein modification of gold nanoparticles alters their cellular uptake at the axonal terminal and activates fast retrograde transport. Trajectory analysis of individual endosomes carrying the nanoparticles reveals a run-and-pause pattern along the axon with endosomes carrying WGA-HRP-conjugated gold nanoparticles exhibiting longer run duration and faster instantaneous velocity than those carrying nonconjugated nanoparticles. The results offer a mechanistic explanation of the different axonal transport dynamics as well as a cell-based functional assay of neuron-targeted nanoparticles with the goal of developing BBB-bypassing nanomedicine for the treatment of nervous system disorders.

Single-molecule imaging of stochastic interactions that drive dynein activation and cargo movement in cells.

Cytoplasmic dynein 1 (dynein) is the primary minus end-directed motor protein in most eukaryotic cells. Dynein remains in an inactive conformation until the formation of a tripartite complex comprising dynein, its regulator dynactin, and a cargo adaptor. How this process of dynein activation occurs is unclear since it entails the formation of a three-protein complex inside the crowded environs of a cell. Here, we employed live-cell, single-molecule imaging to visualize and track fluorescently tagged dynein. First, we observed that only ∼30% of dynein molecules that bound to the microtubule (MT) engaged in minus end-directed movement, and that too for a short duration of ∼0.6 s. Next, using high-resolution imaging in live and fixed cells and using correlative light and electron microscopy, we discovered that dynactin and endosomal cargo remained in proximity to each other and to MTs. We then employed two-color imaging to visualize cargo movement effected by single motor binding. Finally, we performed long-term imaging to show that short movements are sufficient to drive cargo to the perinuclear region of the cell. Taken together, we discovered a search mechanism that is facilitated by dynein's frequent MT binding-unbinding kinetics: (i) in a futile event when dynein does not encounter cargo anchored in proximity to the MT, dynein dissociates and diffuses into the cytoplasm, (ii) when dynein encounters cargo and dynactin upon MT binding, it moves cargo in a short run. Several of these short runs are undertaken in succession for long-range directed movement. In conclusion, we demonstrate that dynein activation and cargo capture are coupled in a step that relies on the reduction of dimensionality to enable minus end-directed transport in cellulo and that complex cargo behavior emerges from stochastic motor-cargo interactions.

Endosomal sorting sorted - motors, adaptors and lessons from in vitro and cellular studies.

Motor proteins are key players in exerting spatiotemporal control over the intracellular location of membrane-bound compartments, including endosomes containing cargo. In this Review, we focus on how motors and their cargo adaptors regulate positioning of cargoes from the earliest stages of endocytosis and through the two main intracellular itineraries: (1) degradation at the lysosome or (2) recycling back to the plasma membrane. In vitro and cellular (in vivo) studies on cargo transport thus far have typically focussed independently on either the motor proteins and adaptors, or membrane trafficking. Here, we will discuss recent studies to highlight what is known about the regulation of endosomal vesicle positioning and transport by motors and cargo adaptors. We also emphasise that in vitro and cellular studies are often performed at different scales, from single molecules to whole organelles, with the aim to provide a perspective on the unified principles of motor-driven cargo trafficking in living cells that can be learned from these differing scales.

Microtubule-mitochondrial attachment facilitates cell division symmetry and mitochondrial partitioning in fission yeast.

Association with microtubules inhibits the fission of mitochondria in Schizosaccharomyces pombe. Here, we show that this attachment of mitochondria to microtubules is an important cell-intrinsic factor in determining cell division symmetry. By comparing mutant cells that exhibited enhanced attachment and no attachment of mitochondria to microtubules (Dnm1Δ and Mmb1Δ, respectively), we show that microtubules in these mutants displayed aberrant dynamics compared to wild-type cells, which resulted in errors in nuclear positioning. This translated to cell division asymmetry in a significant proportion of both Dnm1Δ and Mmb1Δ cells. Asymmetric division in Dnm1Δ and Mmb1Δ cells resulted in unequal distribution of mitochondria, with the daughter cell that received more mitochondria growing faster than the other daughter cell. Taken together, we show the existence of homeostatic feedback controls between mitochondria and microtubules in fission yeast, which directly influence mitochondrial partitioning and, thereby, cell growth. This article has an associated First Person interview with the first author of the paper.

Visualization of Single Dynein Molecules in Mammalian Cells.

In vitro single-molecule imaging experiments have provided insight into the stepping behavior, force production, and activation of several molecular motors. However, due to the difficulty in visualizing single molecules of motor proteins in vivo, the physiological function and regulation of motors at the single-molecule level have not been studied widely. Here, we describe how highly inclined and laminated optical sheet (HILO) microscopy can be adapted to visualize single molecules of the motor protein cytoplasmic dynein-1 in mammalian cells with high signal-to-noise ratio and temporal resolution.

Mitochondrial movers and shapers: Recent insights into regulators of fission, fusion and transport.

Mitochondria are highly dynamic organelles that undergo rapid morphological adaptations influencing their number, transport, cellular distribution, and function, which in turn facilitate the integration of mitochondrial function with physiological changes in the cell. These mitochondrial dynamics are dependent on tightly regulated processes such as fission, fusion, and attachment to the cytoskeleton, and their defects are observed in various pathophysiological conditions including cancer, cardiovascular disease, and neurodegeneration. Various studies over the years have identified key molecular players and uncovered the mechanisms that mediate and regulate these processes and have highlighted their complexity and context-specificity. This review focuses on the recent studies that have contributed to the understanding of processes that influence mitochondrial morphology including fission, fusion, and transport in the cell.

Coupling of mitochondrial population evolution to microtubule dynamics in fission yeast cells: a kinetic Monte Carlo study.

Mitochondrial populations in cells are maintained by cycles of fission and fusion events. Perturbation of this balance has been observed in several diseases such as cancer and neurodegeneration. In fission yeast cells, the association of mitochondria with microtubules inhibits mitochondrial fission [Mehta et al., J. Biol. Chem., 2019, 294, 3385], illustrating the intricate coupling between mitochondria and the dynamic population of microtubules within the cell. In order to understand this coupling, we carried out kinetic Monte Carlo (KMC) simulations to predict the evolution of mitochondrial size distributions for different cases; wild-type cells, cells with short and long microtubules, and cells without microtubules. Comparisons are made with mitochondrial distributions reported in experiments with fission yeast cells. Using experimentally determined mitochondrial fission and fusion frequencies, simulations implemented without the coupling of microtubule dynamics predicted an increase in the mean number of mitochondria, equilibrating within 50 s. The mitochondrial length distribution in these models also showed a higher occurrence of shorter mitochondria, implying a greater tendency for fission, similar to the scenario observed in the absence of microtubules and cells with short microtubules. Interestingly, this resulted in overestimating the mean number of mitochondria and underestimating mitochondrial lengths in cells with wild-type and long microtubules. However, coupling mitochondria's fission and fusion events to the microtubule dynamics effectively captured the mitochondrial number and size distributions in wild-type and cells with long microtubules. Thus, the model provides greater physical insight into the temporal evolution of mitochondrial populations in different microtubule environments, allowing one to study both the short-time evolution as observed in the experiments (<5 minutes) as well as their transition towards a steady-state (>15 minutes). Our study illustrates the critical role of microtubules in mitochondrial dynamics and coupling microtubule growth and shrinkage dynamics is critical to predicting the evolution of mitochondrial populations within the cell.

The story of science: successes, failures, luck, privilege, and bias.

I am incredibly honored to receive the 2021 WICB Junior Award for Excellence in Research in WICB's golden jubilee year. In this essay, I traverse my scientific journey starting with my PhD, highlighting the highs and the lows and how these intersect with luck, privilege, and bias.

Mitochondrial dynamics, positioning and function mediated by cytoskeletal interactions.

The ability of a mitochondrion to undergo fission and fusion, and to be transported and localized within a cell are central not just to proper functioning of mitochondria, but also to that of the cell. The cytoskeletal filaments, namely microtubules, F-actin and intermediate filaments, have emerged as prime movers in these dynamic mitochondrial shape and position transitions. In this review, we explore the complex relationship between the cytoskeleton and the mitochondrion, by delving into: (i) how the cytoskeleton helps shape mitochondria via fission and fusion events, (ii) how the cytoskeleton facilitates the translocation and anchoring of mitochondria with the activity of motor proteins, and (iii) how these changes in form and position of mitochondria translate into functioning of the cell.

Meeting report - the Microtubules, Motors, Transport and Trafficking (M2T2) 2019 meeting.

The Molecular Motors, Transport and Trafficking (M2T2) meeting serves as a platform for both Indian and global scientists working on the cytoskeleton, cytoskeletal motors and membrane trafficking to gather and discuss the latest developments in the field. The 2019 edition of the meeting, held from 18-20 October at the National Brain Research Centre (NBRC), Manesar, India and organised by Mahak Sharma (Indian Institute of Science Education and Research, Mohali) and Anindya Ghosh Roy (NBRC), was witness to stimulating research on a range of topics related to the cytoskeleton, including cytoskeletal organization, motor protein function and regulation, mechanical forces and vesicular transport, and trafficking in health and disease.

Role of Dynactin in the Intracellular Localization and Activation of Cytoplasmic Dynein.

Cytoplasmic dynein, the major minus end-directed motor protein in several cell types, transports a variety of intracellular cargo upon forming a processive tripartite complex with its activator dynactin and cargo adaptors such as Hook3 and BicD2. Our current understanding of dynein regulation stems from a combination of in vivo studies of cargo movement upon perturbation of dynein activity, in vitro single-molecule experiments, and cryo-electron microscopy studies of dynein structure and its interaction with dynactin and cargo adaptors. In this Perspective, we first consolidate data from recent publications to understand how perturbations to the dynein-dynactin interaction and dynactin's in vivo localization alter the behavior of dynein-driven cargo transport in a cell type- and experimental condition-specific manner. In addition, we touch upon results from in vivo and in vitro studies to elucidate how dynein's interaction with dynactin and cargo adaptors activates dynein and enhances its processivity. Finally, we propose questions that need to be addressed in the future with appropriate experimental designs so as to improve our understanding of the spatiotemporal regulation of dynein's function in the context of the distribution and dynamics of dynactin in living cells.

Cortical tethering of mitochondria by the anchor protein Mcp5 enables uniparental inheritance.

During sexual reproduction in eukaryotes, processes such as active degradation and dilution of paternal mitochondria ensure maternal mitochondrial inheritance. In the isogamous organism fission yeast, we employed high-resolution fluorescence microscopy to visualize mitochondrial inheritance during meiosis by differentially labeling mitochondria of the two parental cells. Remarkably, mitochondria, and thereby mitochondrial DNA from the parental cells, did not mix upon zygote formation but remained segregated at the poles by attaching to clusters of the anchor protein Mcp5 via its coiled-coil domain. We observed that this tethering of parental mitochondria to the poles results in uniparental inheritance of mitochondria, wherein two of the four spores formed subsequently contained mitochondria from one parent and the other spores contained mitochondria from the other parent. Further, the presence of dynein on an Mcp5 cluster precluded the attachment of mitochondria to the same cluster. Taken together, we reveal a distinct mechanism that achieves uniparental inheritance by segregation of parental mitochondria.

Association of mitochondria with microtubules inhibits mitochondrial fission by precluding assembly of the fission protein Dnm1.

Mitochondria are organized as tubular networks in the cell and undergo fission and fusion. Although several of the molecular players involved in mediating mitochondrial dynamics have been identified, the precise cellular cues that initiate mitochondrial fission or fusion remain largely unknown. In fission yeast (Schizosaccharomyces pombe), mitochondria are organized along microtubule bundles. Here, we employed deletions of kinesin-like proteins to perturb microtubule dynamics and used high-resolution and time-lapse fluorescence microscopy, revealing that mitochondrial lengths mimic microtubule lengths. Furthermore, we determined that compared with WT cells, mutant cells with long microtubules exhibit fewer mitochondria, and mutant cells with short microtubules have an increased number of mitochondria because of reduced mitochondrial fission in the former and elevated fission in the latter. Correspondingly, upon onset of closed mitosis in fission yeast, wherein interphase microtubules assemble to form the spindle within the nucleus, we observed increased mitochondrial fission. We found that the consequent rise in the mitochondrial copy number is necessary to reduce partitioning errors during independent segregation of mitochondria between daughter cells. We also discovered that the association of mitochondria with microtubules physically impedes the assembly of the fission protein Dnm1 around mitochondria, resulting in inhibition of mitochondrial fission. Taken together, we demonstrate a mechanism for the regulation of mitochondrial fission that is dictated by the interaction between mitochondria and the microtubule cytoskeleton.

Quantification of Mitochondrial Dynamics in Fission Yeast.

Mitochondria are double-membraned organelles responsible for several functions in the cell including energy production, calcium signaling, and cellular metabolism. An equilibrium between fission and fusion events of mitochondria is required for their proper functioning. Mitochondrial morphologies have been quantified in yeast using image processing modules such as MitoGraph and MitoLoc. However, the dynamics of mitochondrial fission and fusion have not been analyzed in these methods. Here, we present a method for measuring mitochondrial morphologies, as well as estimation of fission and fusion frequencies of mitochondria in individual fission yeast cells whose mitochondria are fluorescently-tagged or stained. The latter relies on counting of individual mitochondria upon signal filtering in each frame of a time-lapse. Taken together, we present a simple protocol for analyzing mitochondrial dynamics, which can easily be adopted to other model systems.

Fission yeast myosin I facilitates PI(4,5)P2-mediated anchoring of cytoplasmic dynein to the cortex.

Several key processes in the cell, such as vesicle transport and spindle positioning, are mediated by the motor protein cytoplasmic dynein, which produces force on the microtubule. For the functions that require movement of the centrosome and the associated nuclear material, dynein needs to have a stable attachment at the cell cortex. In fission yeast, Mcp5 is the anchor protein of dynein and is required for the oscillations of the horsetail nucleus during meiotic prophase. Although the role of Mcp5 in anchoring dynein to the cortex has been identified, it is unknown how Mcp5 associates with the membrane as well as the importance of the underlying attachment to the nuclear oscillations. Here, we set out to quantify Mcp5 organization and identify the binding partner of Mcp5 at the membrane. We used confocal and total internal reflection fluorescence microscopy to count the number of Mcp5 foci and the number of Mcp5 molecules in an individual focus. Further, we quantified the localization pattern of Mcp5 in fission yeast zygotes and show by perturbation of phosphatidylinositol 4-phosphate 5-kinase that Mcp5 binds to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Remarkably, we discovered that the myosin I protein in fission yeast, Myo1, which is required for organization of sterol-rich domains in the cell membrane, facilitates the localization of Mcp5 and that of cytoplasmic dynein on the membrane. Finally, we demonstrate that Myo1-facilitated association of Mcp5 and dynein to the membrane determines the dynamics of nuclear oscillations and, in essence, dynein activity.