A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2.

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

Lis1 relieves cytoplasmic dynein-1 autoinhibition by acting as a molecular wedge.

Cytoplasmic dynein-1 transports intracellular cargo towards microtubule minus ends. Dynein is autoinhibited and undergoes conformational changes to form an active complex that consists of one or two dynein dimers, the dynactin complex, and activating adapter(s). The Lissencephaly 1 gene, LIS1, is genetically linked to the dynein pathway from fungi to mammals and is mutated in people with the neurodevelopmental disease lissencephaly. Lis1 is required for active dynein complexes to form, but how it enables this is unclear. Here, we present a structure of two yeast dynein motor domains with two Lis1 dimers wedged in-between. The contact sites between dynein and Lis1 in this structure, termed 'Chi,' are required for Lis1's regulation of dynein in Saccharomyces cerevisiae in vivo and the formation of active human dynein-dynactin-activating adapter complexes in vitro. We propose that this structure represents an intermediate in dynein's activation pathway, revealing how Lis1 relieves dynein's autoinhibited state.

Structural basis for cytoplasmic dynein-1 regulation by Lis1.

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein's motor domain, and by doing so alters dynein's mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1's ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1's ability to form fully active human dynein complexes and to regulate yeast dynein's mechanochemistry and in vivo function.

A Septin Double Ring Controls the Spatiotemporal Organization of the ESCRT Machinery in Cytokinetic Abscission.

Abscission is the terminal step of mitosis that physically separates two daughter cells [1, 2]. Abscission requires the endocytic sorting complex required for transport (ESCRT), a molecular machinery of multiple subcomplexes (ESCRT-I/II/III) that promotes membrane remodeling and scission [3-5]. Recruitment of ESCRT-I/II complexes to the midbody of telophase cells initiates ESCRT-III assembly into two rings, which subsequently expand into helices and spirals that narrow down to the incipient site of abscission [6-8]. ESCRT-III assembly is highly dynamic and spatiotemporally ordered, but the underlying mechanisms are poorly understood. Here, we report that, after cleavage furrow closure, septins form a membrane-bound double ring that controls the organization and function of ESCRT-III. The septin double ring demarcates the sites of ESCRT-III assembly into rings and disassembles before ESCRT-III rings expand into helices and spirals. We show that septin 9 (SEPT9) depletion, which abrogates abscission, impairs recruitment of VPS25 (ESCRT-II) and CHMP6 (ESCRT-III). Strikingly, ESCRT-III subunits (CHMP4B and CHMP2A/B) accumulate to the midbody, but they are highly disorganized, failing to form symmetric rings and to expand laterally into the cone-shaped helices and spirals of abscission. We found that SEPT9 interacts directly with the ubiquitin E2 variant (UEV) domain of ESCRT-I protein TSG101 through two N-terminal PTAP motifs, which are required for the recruitment of VPS25 and CHMP6, and the spatial organization of ESCRT-III (CHMP4B and CHMP2B) into functional rings. These results reveal that septins function in the ESCRT-I-ESCRT-II-CHMP6 pathway of ESCRT-III assembly and provide a framework for the spatiotemporal control of the ESCRT machinery of cytokinetic abscission.

Doing science in difficult socioeconomic circumstances.

Doing scientific research can be a challenging, but creative and rewarding career. However, the challenge of doing research is orders of magnitude more difficult for scientists lacking access to the necessary resources. Resource-scarce environments are quite common worldwide and are highly contingent on the financial and social climate of one's location. Through the experiences of four scientists, this Perspective explores the challenges associated with doing research in disadvantaged socioeconomic circumstances. By reflecting on the humble beginnings of these scientists, we examine the motivation to stay in or leave one's home country, and how hardships can be leveraged to cultivate passionate and productive researchers.

Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin.

Neuronal function requires axon-dendrite membrane polarity, which depends on sorting of membrane traffic during entry into axons. Due to a microtubule network of mixed polarity, dendrites receive vesicles from the cell body without apparent capacity for directional sorting. We found that, during entry into dendrites, axonally destined cargos move with a retrograde bias toward the cell body, while dendritically destined cargos are biased in the anterograde direction. A microtubule-associated septin (SEPT9), which localizes specifically in dendrites, impedes axonal cargo of kinesin-1/KIF5 and boosts kinesin-3/KIF1 motor cargo further into dendrites. In neurons and in vitro single-molecule motility assays, SEPT9 suppresses kinesin-1/KIF5 and enhances kinesin-3/KIF1 in a manner that depends on a lysine-rich loop of the kinesin motor domain. This differential regulation impacts partitioning of neuronal membrane proteins into axons-dendrites. Thus, polarized membrane traffic requires sorting during entry into dendrites by a septin-mediated mechanism that bestows directional bias on microtubules of mixed orientation.

Septin 9 interacts with kinesin KIF17 and interferes with the mechanism of NMDA receptor cargo binding and transport.

Intracellular transport involves the regulation of microtubule motor interactions with cargo, but the underlying mechanisms are not well understood. Septins are membrane- and microtubule-binding proteins that assemble into filamentous, scaffold-like structures. Septins are implicated in microtubule-dependent transport, but their roles are unknown. Here we describe a novel interaction between KIF17, a kinesin 2 family motor, and septin 9 (SEPT9). We show that SEPT9 associates directly with the C-terminal tail of KIF17 and interacts preferentially with the extended cargo-binding conformation of KIF17. In developing rat hippocampal neurons, SEPT9 partially colocalizes and comigrates with KIF17. We show that SEPT9 interacts with the KIF17 tail domain that associates with mLin-10/Mint1, a cargo adaptor/scaffold protein, which underlies the mechanism of KIF17 binding to the NMDA receptor subunit 2B (NR2B). Significantly, SEPT9 interferes with binding of the PDZ1 domain of mLin-10/Mint1 to KIF17 and thereby down-regulates NR2B transport into the dendrites of hippocampal neurons. Measurements of KIF17 motility in live neurons show that SEPT9 does not affect the microtubule-dependent motility of KIF17. These results provide the first evidence of an interaction between septins and a nonmitotic kinesin and suggest that SEPT9 modulates the interactions of KIF17 with membrane cargo.