Light Intermediate Chain 1 Research Articles

A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.

A transient helix in the disordered region of dynein light intermediate chain links the motor to structurally diverse adaptors for cargo transport.

All animal cells use the motor cytoplasmic dynein 1 (dynein) to transport diverse cargo toward microtubule minus ends and to organize and position microtubule arrays such as the mitotic spindle. Cargo-specific adaptors engage with dynein to recruit and activate the motor, but the molecular mechanisms remain incompletely understood. Here, we use structural and dynamic nuclear magnetic resonance (NMR) analysis to demonstrate that the C-terminal region of human dynein light intermediate chain 1 (LIC1) is intrinsically disordered and contains two short conserved segments with helical propensity. NMR titration experiments reveal that the first helical segment (helix 1) constitutes the main interaction site for the adaptors Spindly (SPDL1), bicaudal D homolog 2 (BICD2), and Hook homolog 3 (HOOK3). In vitro binding assays show that helix 1, but not helix 2, is essential in both LIC1 and LIC2 for binding to SPDL1, BICD2, HOOK3, RAB-interacting lysosomal protein (RILP), RAB11 family-interacting protein 3 (RAB11FIP3), ninein (NIN), and trafficking kinesin-binding protein 1 (TRAK1). Helix 1 is sufficient to bind RILP, whereas other adaptors require additional segments preceding helix 1 for efficient binding. Point mutations in the C-terminal helix 1 of Caenorhabditis elegans LIC, introduced by genome editing, severely affect development, locomotion, and life span of the animal and disrupt the distribution and transport kinetics of membrane cargo in axons of mechanosensory neurons, identical to what is observed when the entire LIC C-terminal region is deleted. Deletion of the C-terminal helix 2 delays dynein-dependent spindle positioning in the one-cell embryo but overall does not significantly perturb dynein function. We conclude that helix 1 in the intrinsically disordered region of LIC provides a conserved link between dynein and structurally diverse cargo adaptor families that is critical for dynein function in vivo.

A conserved interaction of the dynein light intermediate chain with dynein-dynactin effectors necessary for processivity

Cytoplasmic dynein is the major minus-end-directed microtubule-based motor in cells. Dynein processivity and cargo selectivity depend on cargo-specific effectors that, while generally unrelated, share the ability to interact with dynein and dynactin to form processive dynein–dynactin-effector complexes. How this is achieved is poorly understood. Here, we identify a conserved region of the dynein Light Intermediate Chain 1 (LIC1) that mediates interactions with unrelated dynein–dynactin effectors. Quantitative binding studies map these interactions to a conserved helix within LIC1 and to N-terminal fragments of Hook1, Hook3, BICD2, and Spindly. A structure of the LIC1 helix bound to the N-terminal Hook domain reveals a conformational change that creates a hydrophobic cleft for binding of the LIC1 helix. The LIC1 helix competitively inhibits processive dynein–dynactin-effector motility in vitro, whereas structure-inspired mutations in this helix impair lysosomal positioning in cells. The results reveal a conserved mechanism of effector interaction with dynein–dynactin necessary for processive motility.

Assembly and activation of dynein-dynactin by the cargo adaptor protein Hook3.

Metazoan cytoplasmic dynein moves processively along microtubules with the aid of dynactin and an adaptor protein that joins dynein and dynactin into a stable ternary complex. Here, we examined how Hook3, a cargo adaptor involved in Golgi and endosome transport, forms a motile dynein-dynactin complex. We show that the conserved Hook domain interacts directly with the dynein light intermediate chain 1 (LIC1). By solving the crystal structure of the Hook domain and using structure-based mutagenesis, we identify two conserved surface residues that are each critical for LIC1 binding. Hook proteins with mutations in these residues fail to form a stable dynein-dynactin complex, revealing a crucial role for LIC1 in this interaction. We also identify a region of Hook3 specifically required for an allosteric activation of processive motility. Our work reveals the structural details of Hook3's interaction with dynein and offers insight into how cargo adaptors form processive dynein-dynactin motor complexes.

PKA-dependent dynein switching from lysosomes to adenovirus: a novel form of host-virus competition.

Cytoplasmic dynein is responsible for transport of several viruses to the nucleus. Adenovirus recruits dynein directly. Transport depends on virus-induced activation of protein kinase A (PKA) and other cellular protein kinases, whose roles in infection are poorly understood. We find that PKA phosphorylates cytoplasmic dynein at a novel site in light intermediate chain 1 (LIC1) that is essential for dynein binding to the hexon capsid subunit and for virus motility. Surprisingly, the same LIC1 modification induces a slow, but specific, dispersal of lysosomes (lyso)/late endosomes (LEs) that is mediated by inhibition of a newly identified LIC1 interaction with the RILP (Rab7-interacting lysosomal protein). These results identify an organelle-specific dynein regulatory modification that adenovirus uses for its own transport. PKA-mediated LIC1 phosphorylation causes only partial lyso/LE dispersal, suggesting a role for additional, parallel mechanisms for dynein recruitment to lyso/LEs. This arrangement provides a novel means to fine tune transport of these organelles in response to infection as well as to developmental and physiological cues.

Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.

The Small GTPase Rab4A Interacts with the Central Region of Cytoplasmic Dynein Light Intermediate Chain-1

Rab4 belongs to the Rab family of small GTPases involved in the regulation of intracellular transport, and has been localized to early endosomes. We have employed the yeast two-hybrid system to identify proteins that specifically interact with Rab4AQ67L, a GTPase-deficient mutant form of Rab4A. Screening a mouse embryo cDNA library identified a clone (M449) that interacted with Rab4A in a nucleotide-dependent fashion. Data base searches identified this clone as the mouse cytoplasmic dynein light intermediate chain-1 (LIC-1). Based on this finding, the full-length equivalent human cytoplasmic dynein LIC-1 was isolated by PCR. When Rab4A was overexpressed together with either M449 or dynein LIC-1 in HeLa cells, the proteins were found to colocalize in the perinuclear region. We characterize the localization of both overexpressed human dynein LIC-1 and the endogenous protein with respect to microtubules and show that it concentrates to the microtubule-organizing center and mitotic spindle. Additionally, GFPRab4A endosomes localize to microtubules and are redistributed by nocodazole treatment. This is the first described interaction between cytoplasmic dynein, a retrograde motor protein, and a Rab protein.