Conservation and divergence within the clathrin interactome of <i>Trypanosoma cruzi</i>

Trypanosomatids are parasitic protozoa with a significant burden on human health. African and American trypanosomes are causative agents of Nagana and Chagas disease respectively, and speciated about 300 million years ago. These parasites have highly distinct life cycles, pathologies, transmission strategies and surface proteomes, being dominated by the variant surface glycoprotein (African) or mucins (American) respectively. In African trypanosomes clathrin-mediated trafficking is responsible for endocytosis and post-Golgi transport, with several mechanistic aspects distinct from higher organisms. Using clathrin light chain (TcCLC) and EpsinR (TcEpsinR) as affinity handles, we identified candidate clathrin-associated proteins (CAPs) in Trypanosoma cruzi; the cohort includes orthologs of many proteins known to mediate vesicle trafficking, but significantly not the AP-2 adaptor complex. Several trypanosome-specific proteins common with African trypanosomes, were also identified. Fluorescence microscopy revealed localisations for TcEpsinR, TcCLC and TcCHC at the posterior region of trypomastigote cells, coincident with the flagellar pocket and Golgi apparatus. These data provide the first systematic analysis of clathrin-mediated trafficking in T. cruzi, allowing comparison between protein cohorts and other trypanosomes and also suggest that clathrin trafficking in at least some life stages of T. cruzi may be AP-2-independent

Quantitative Mass Spectrometry Analysis Reveals Similar Substrate Consensus Motif for Human Mps1 Kinase and Plk1

Background Members of the Mps1 kinase family play an essential and evolutionarily conserved role in the spindle assembly checkpoint (SAC), a surveillance mechanism that ensures accurate chromosome segregation during mitosis. Human Mps1 (hMps1) is highly phosphorylated during mitosis and many phosphorylation sites have been identified. However, the upstream kinases responsible for these phosphorylations are not presently known. Methodology/Principal Findings Here, we identify 29 in vivo phosphorylation sites in hMps1. While in vivo analyses indicate that Aurora B and hMps1 activity are required for mitotic hyper-phosphorylation of hMps1, in vitro kinase assays show that Cdk1, MAPK, Plk1 and hMps1 itself can directly phosphorylate hMps1. Although Aurora B poorly phosphorylates hMps1 in vitro, it positively regulates the localization of Mps1 to kinetochores in vivo. Most importantly, quantitative mass spectrometry analysis demonstrates that at least 12 sites within hMps1 can be attributed to autophosphorylation. Remarkably, these hMps1 autophosphorylation sites closely resemble the consensus motif of Plk1, demonstrating that these two mitotic kinases share a similar substrate consensus. Conclusions/Significance hMps1 kinase is regulated by Aurora B kinase and its autophosphorylation. Analysis on hMps1 autophosphorylation sites demonstrates that hMps1 has a substrate preference similar to Plk1 kinase

Mps1Mph1 kinase phosphorylates Mad3 to inhibit Cdc20Slp1-APC/C and maintain spindle checkpoint arrests

<div><p>The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1^Mph1 kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1^Mph1 kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors <i>in vitro</i>, demonstrating that Mad3p modification can directly influence Cdc20^Slp1-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1^Mph1 kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.</p></div

Control of the spindle checkpoint by lateral kinetochore attachment and limited Mad1 recruitment

We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay

How the kinetochore couples microtubule force and centromere stretch to move chromosomes

The Ndc80 complex (Ndc80, Nuf2, Spc24, Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus-ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule and MAP binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus-end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule binding interactions with Ndc80 and Dam1 complexes

Design of a hyperstable 60-subunit protein icosahedron

The icosahedron and the dodecahedron are the largest of the Platonic solids, and icosahedral protein structures are widely utilized in biological systems for packaging and transport(1,2). There has been considerable interest in repurposing such structures(3–5), for example, virus-like particles for the targeted delivery and vaccine design. The ability to design proteins that self assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein 'containers' that could exhibit properties custom-made for various applications. In this manuscript, we describe the computational design of an icosahedral nano-cage that self-assembles from trimeric building blocks. Electron microscopy images of the designed protein expressed in E. coli reveals a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 M guanidine hydrochloride at up to 80 °C, and undergo extremely abrupt, but reversible, disassembly between 2 M and 2.25 M guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of superfolder GFP can be fused to each of the 60 subunits to create highly fluorescent standard candles for light microscopy, and a designed protein pentamer can be placed in the center of each of the twenty pentameric faces to potentially gate macromolecule access to the nanocage interior. Such robust designed nanocages should have considerable utility for targeted drug delivery(6), vaccine design(7), and synthetic biology(8)