Kevin C. Slep

The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction.

Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin–afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila melanogaster’s afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. We completely removed Cno from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin–catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis.

Structural and mechanistic insights into microtubule end-binding proteins

Recent experiments reconstituting microtubule plus end tracking activity coupled with structural determination of microtubule plus end domains and plus end complexes are revealing the hierarchy, regulatory features, and potential mechanisms of plus end tracking proteins. Primary plus end tracking proteins include EB1 and XMAP215, while a host of secondary, EB1-dependent plus end proteins have been identified and characterized, including CLIP-170 and SKIP-motif proteins. Single molecule in vitro reconstitution assays show that XMAP215 is a processive polymerases that drives tubulin polymerization. Analysis of the EB1-microtubule interaction indicates EB1 actively promotes A-form microtubule lattice growth and rapidly exchanges with subsecond dwell times.

Molecular architecture of Gαo and the structural basis for RGS16-mediated deactivation

Heterotrimeric G proteins relay extracellular cues from heptahelical transmembrane receptors to downstream effector molecules. Composed of an α subunit with intrinsic GTPase activity and a βγ heterodimer, the trimeric complex dissociates upon receptor-mediated nucleotide exchange on the α subunit, enabling each component to engage downstream effector targets for either activation or inhibition as dictated in a particular pathway. To mitigate excessive effector engagement and concomitant signal transmission, the Gα subunits intrinsic activation timer (the rate of GTP hydrolysis) is regulated spatially and temporally by a class of GTPase accelerating proteins (GAPs) known as the regulator of G protein signaling (RGS) family. The array of G protein-coupled receptors, Gα subunits, RGS proteins and downstream effectors in mammalian systems is vast. Understanding the molecular determinants of specificity is critical for a comprehensive mapping of the G protein system. Here, we present the 2.9 Å crystal structure of the enigmatic, neuronal G protein Gαo in the GTP hydrolytic transition state, complexed with RGS16. Comparison with the 1.89 Å structure of apo-RGS16, also presented here, reveals plasticity upon Gαo binding, the determinants for GAP activity, and the structurally unique features of Gαo that likely distinguish it physiologically from other members of the larger Gαi family, affording insight to receptor, GAP and effector specificity.

The role of TOG domains in microtubule plus end dynamics.

The XMAP215 (Xenopus microtubule-associated protein 215) and CLASP [CLIP-170 (cytoskeletal linker protein 170) associated protein] microtubule plus end tracking families play central roles in the regulation of interphase microtubule dynamics and the proper formation of mitotic spindle architecture and flux. XMAP215 members comprise N-terminally-arrayed hexa-HEAT (huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor) repeats known as TOG (tumour overexpressed gene) domains. Higher eukaryotic XMAP215 members are monomeric and have five TOG domains. Yeast counterparts are dimeric and have two TOG domains. Structure determination of the TOG domain reveals that the six HEAT repeats are aligned to form an oblong scaffold. The TOG domain face composed of intra-HEAT loops forms a contiguous, conserved tubulin-binding surface. Nested within the conserved intra-HEAT loop 1 is an invariant, signature, surface-exposed tryptophan residue that is a prime determinant in the TOG domain-tubulin interaction. The arrayed organization of TOG domains is critical for the processive mechanism of XMAP215, indicative that multiple tubulin/microtubule-binding sites are required for plus end tracking activity. The CLASP family has been annotated as containing a single N-terminal TOG domain. Using XMAP215 TOG domain structure determinants as a metric to analyse CLASP sequence, it is anticipated that CLASP contains two additional cryptic TOGL (TOG-like) domains. The presence of additional TOGL domains implicates CLASP as an ancient XMAP215 relative that uses a similar, multi-TOG-based mechanism to processively track microtubule ends.

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

An in vivo structure–function analysis of the Drosophila homologue Mini spindles (Msps) identified novel domains that are necessary for the interplay between the conserved TOG domains and inter-TOG microtubule (MT) binding that underlies the ability of Msps to promote MT dynamic instability.

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

The Asterless N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas its C terminus stabilizes Plk4 during mitosis.

The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array

Structures of Drosophila Msps TOG4 and human ch-TOG TOG4 are presented. TOG4 departs from the other TOG structures and predicts novel α-tubulin engagement. Whereas TOG domains across the array have different tubulin-binding properties, cellular studies show that a fully functional TOG array is required for microtubule polymerase activity.

The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: Structural and single-molecule characterization

Studying the role of accessory chains in dynein single-molecule motility shows that the dynein light chain (LC) and intermediate chain (IC) promote motor dimerization, increase velocity, and potentiate processivity. The crystal structure of the yeast LC–IC complex is determined, and the interaction is biochemically characterized.

Crescerin uses a TOG domain array to regulate microtubules in the primary cilium

Primary cilia are critical organelles involved in development, sensation, and signaling. Crescerin, a conserved protein family in ciliated and flagellated eukaryotes, uses a TOG domain array with tubulin polymerization activity to regulate cilia microtubules and facilitate proper cilia length, ultrastructure, and function.

The Mechanism of Dynein Light Chain LC8-mediated Oligomerization of the Ana2 Centriole Duplication Factor

Background: Ana2 is a conserved centriole duplication factor involved in nascent centriole biogenesis. Results: Two sites in the central domain of Ana2 (Ana2M) bind LC8 and form an Ana2M4-LC88 complex. Conclusion: LC8 potentiates Ana2 tetramerization. Significance: LC8-potentiated Ana2 tetramerization is expected to increase the avidity of Ana2 for centriole factors, including Sas-6, and may drive binding factor oligomerization. Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila anastral spindle-2 (Ana2) is a conserved centriole duplication factor. Although recent work has demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. Here we examine the Ana2-LC8 interaction and map two LC8-binding sites within the central region of Ana2, Ana2M (residues 156–251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 μm), whereas site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 μm). Both LC8-binding sites flank a predicted ∼34-residue α-helix. We present two independent atomic structures of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form β-strands that extend a central composite LC8 β-sandwich. LC8 recognizes the signature TQT motif in the first LC8 binding site of Ana2, forming extensive van der Waals contacts and hydrogen bonding with the peptide, whereas the Ana2 site 2 TQC motif forms a uniquely extended β-strand, not observed in other dynein light chain-target complexes. Size exclusion chromatography coupled with multiangle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization probably enhances Ana2 avidity for centriole-binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis.