Genome-scale functional genomics screening highlights genes impacting protein fucosylation in Chinese hamster ovary cells.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

Microtubule-Based Transport and the Distribution, Tethering, and Organization of Organelles.

SUMMARYMicrotubules provide long tracks along which a broad range of organelles and vesicles are transported by kinesin and dynein motors. Motor protein complexes also tether cargoes to cytoskeletal filaments, helping facilitate their interaction and communication. The generation of biochemically distinct microtubule subpopulations allows subsets of motors to recognize a given microtubule identity, allowing further organization within the cytoplasm. Both transport and tethering are spatiotemporally regulated through multiple modes, including acute modification of both motor-cargo and motor-track associations by various physiological signals. Strict regulation of intracellular transport is particularly important in specialized cell types such as neurons. Here, we review general mechanisms by which cargo transport is controlled and also highlight examples of transport regulated by multiple mechanisms.

Fat2 and Lar Define a Basally Localized Planar Signaling System Controlling Collective Cell Migration.

Collective migration of epithelial cells underlies diverse tissue-remodeling events, but the mechanisms that coordinate individual cell migratory behaviors for collective movement are largely unknown. Studying the Drosophila follicular epithelium, we show that the cadherin Fat2 and the receptor tyrosine phosphatase Lar function in a planar signaling system that coordinates leading and trailing edge dynamics between neighboring cells. Fat2 signals from each cell's trailing edge to induce leading edge protrusions in the cell behind, in part by stabilizing Lar's localization in these cells. Conversely, Lar signals from each cell's leading edge to stimulate trailing edge retraction in the cell ahead. Fat2/Lar signaling is similar to planar cell polarity signaling in terms of sub-cellular protein localization; however, Fat2/Lar signaling mediates short-range communication between neighboring cells instead of transmitting long-range information across a tissue. This work defines a key mechanism promoting epithelial migration and establishes a different paradigm for planar cell-cell signaling.

The journey of the organelle: teamwork and regulation in intracellular transport.

Specific subsets of biochemical reactions in eukaryotic cells are restricted to individual membrane compartments, or organelles. Cells, therefore, face the monumental task of moving the products of those reactions between individual organelles. Because of the high density of the cytoplasm and the large size of membrane organelles, simple diffusion is grossly insufficient for this task. Proper trafficking between membrane organelles thus relies on cytoskeletal elements and the activity of motor proteins, that act both in transport of membrane compartments and as tethering agents to ensure their proper distribution and to facilitate organelle interactions.

The microtubule-binding protein ensconsin is an essential cofactor of kinesin-1.

Kinesin-1 is a major microtubule motor that drives transport of numerous cellular cargoes toward the plus ends of microtubules. In the cell, kinesin-1 exists primarily in an inactive, autoinhibited state, and motor activation is thought to occur upon binding to cargo through the C terminus. Using RNAi-mediated depletion in Drosophila S2 cells, we demonstrate that kinesin-1 requires ensconsin (MAP7, E-MAP-115), a ubiquitous microtubule-associated protein, for its primary function of organelle transport. We show that ensconsin is required for organelle transport in Drosophila neurons and that Drosophila homozygous for ensconsin gene deletion are unable to survive to adulthood. An ensconsin N-terminal truncation that cannot bind microtubules is sufficient to activate organelle transport by kinesin-1, indicating that this activating domain functions independently of microtubule binding. Interestingly, ens mutant flies retaining expression of this truncation show normal viability. A "hingeless" mutant of kinesin-1, which mimics the active conformation of the motor, does not require ensconsin for transport in S2 cells, suggesting that ensconsin plays a role in relieving autoinhibition of kinesin-1. Together with other recent work, our study suggests that ensconsin is an essential cofactor for all known functions of kinesin-1.

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein.

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.

Statistics of active transport in Xenopus melanophores cells.

The transport of cell cargo, such as organelles and protein complexes in the cytoplasm, is determined by cooperative action of molecular motors stepping along polar cytoskeletal elements. Analysis of transport of individual organelles generated useful information about the properties of the motor proteins and underlying cytoskeletal elements. In this work, for the first time (to our knowledge), we study collective movement of multiple organelles using Xenopus melanophores, pigment cells that translocate several thousand of pigment granules (melanosomes), spherical organelles of a diameter of ∼1 µm. These cells disperse melanosomes in the cytoplasm in response to high cytoplasmic cAMP, while at low cAMP melanosomes cluster at the cell center. Obtained results suggest spatial and temporal organization, characterized by strong correlations between movement of neighboring organelles, with correlation length of ∼4 µm and pair lifetime ∼5 s. Furthermore, velocity statistics revealed strongly non-Gaussian velocity distribution with high velocity tails demonstrating exponential behavior suggestive of strong velocity correlations. Depolymerization of vimentin intermediate filaments using a dominant-negative vimentin mutant or actin with cytochalasin B reduced correlation of behavior of individual particles. Based on our analysis, we concluded that steric repulsion is dominant, but both intermediate filaments and actin microfilaments are involved in dynamic cross-linking organelles in the cytoplasm.

Intracellular transport: ER and mitochondria meet and greet along designated tracks.

A recent study shows that contacts between the endoplasmic reticulum and mitochondria occur preferentially on acetylated microtubules, providing physiological support for the microtubule track selectivity of molecular motors.

Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants.

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function.

Opposite-polarity motors activate one another to trigger cargo transport in live cells.

Intracellular transport is typically bidirectional, consisting of a series of back and forth movements. Kinesin-1 and cytoplasmic dynein require each other for bidirectional transport of intracellular cargo along microtubules; i.e., inhibition or depletion of kinesin-1 abolishes dynein-driven cargo transport and vice versa. Using Drosophila melanogaster S2 cells, we demonstrate that replacement of endogenous kinesin-1 or dynein with an unrelated, peroxisome-targeted motor of the same directionality activates peroxisome transport in the opposite direction. However, motility-deficient versions of motors, which retain the ability to bind microtubules and hydrolyze adenosine triphosphate, do not activate peroxisome motility. Thus, any pair of opposite-polarity motors, provided they move along microtubules, can activate one another. These results demonstrate that mechanical interactions between opposite-polarity motors are necessary and sufficient for bidirectional organelle transport in live cells.

The dynamic properties of intermediate filaments during organelle transport.

Intermediate filament (IF) dynamics during organelle transport and their role in organelle movement were studied using Xenopus laevis melanophores. In these cells, pigment granules (melanosomes) move along microtubules and microfilaments, toward and away from the cell periphery in response to alpha-melanocyte stimulating hormone (alpha-MSH) and melatonin, respectively. In this study we show that melanophores possess a complex network of vimentin IFs which interact with melanosomes. IFs form an intricate, honeycomb-like network that form cages surrounding individual and small clusters of melanosomes, both when they are aggregated and dispersed. Purified melanosome preparations contain a substantial amount of vimentin, suggesting that melanosomes bind to IFs. Analyses of individual melanosome movements in cells with disrupted IF networks show increased movement of granules in both anterograde and retrograde directions, further supporting the notion of a melanosome-IF interaction. Live imaging reveals that IFs, in turn, become highly flexible as melanosomes disperse in response to alpha-MSH. During the height of dispersion there is a marked increase in the rate of fluorescence recovery after photobleaching of GFP-vimentin IFs and an increase in vimentin solubility. These results reveal a dynamic interaction between membrane bound pigment granules and IFs and suggest a role for IFs as modulators of granule movement.