Fabrication of High Aspect Ratio Gold Nanowires within the Microtubule Lumen.

Gold nanowires have great potential use as interconnects in electronic, photonic, and optoelectronic devices. To date, there are various fabrication strategies for gold nanowires, each one associated with particular drawbacks as they utilize high temperatures, toxic chemicals, or expensive compounds to produce nanowires of suboptimal quality. Inspired by nanowire fabrication strategies that used higher-order biopolymer structures as molds for electroless deposition of gold, we here report a strategy for the growth of gold nanowires from seed nanoparticles within the lumen of microtubules. Luminal targeting of seed particles occurs through covalently linked Fab fragments of an antibody recognizing the acetylated lysine 40 on the luminal side of α-tubulin. Gold nanowires grown by electroless deposition within the microtubule lumen exhibit a homogeneous morphology and high aspect ratios with a mean diameter of 20 nm. Our approach is fast, simple, and inexpensive and does not require toxic chemicals or other harsh conditions.

The ALS-Associated FUS (P525L) Variant Does Not Directly Interfere with Microtubule-Dependent Kinesin-1 Motility.

Deficient intracellular transport is a common pathological hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Mutations in the fused-in-sarcoma (FUS) gene are one of the most common genetic causes for familial ALS. Motor neurons carrying a mutation in the nuclear localization sequence of FUS (P525L) show impaired axonal transport of several organelles, suggesting that mislocalized cytoplasmic FUS might directly interfere with the transport machinery. To test this hypothesis, we studied the effect of FUS on kinesin-1 motility in vitro. Using a modified microtubule gliding motility assay on surfaces coated with kinesin-1 motor proteins, we showed that neither recombinant wildtype and P525L FUS variants nor lysates from isogenic ALS-patient-specific iPSC-derived spinal motor neurons expressing those FUS variants significantly affected gliding velocities. We hence conclude that during ALS pathogenesis the initial negative effect of FUS (P525L) on axonal transport is an indirect nature and requires additional factors or mechanisms.

Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles.

Human Kinesin-12 (hKif15) plays a crucial role in assembly and maintenance of the mitotic spindle. These functions of hKif15 are partially redundant with Kinesin-5 (Eg5), which can cross-link and drive the extensile sliding of antiparallel microtubules. Although both motors are known to be tetramers, the functional properties of hKif15 are less well understood. Here we reveal how single or multiple Kif15 motors can cross-link, transport, and focus the plus-ends of intersecting microtubules. During transport, Kif15 motors step simultaneously along both microtubules with relative microtubule transport driven by a velocity differential between motor domain pairs. Remarkably, this differential is affected by the underlying intersection geometry: the differential is low on parallel and extreme on antiparallel microtubules where one motor domain pair becomes immobile. As a result, when intersecting microtubules are antiparallel, canonical transport of one microtubule along the other is allowed because one motor is firmly attached to one microtubule while it is stepping on the other. When intersecting microtubules are parallel, however, Kif15 motors can drive (biased) parallel sliding because the motor simultaneously steps on both microtubules that it cross-links. These microtubule rearrangements will focus microtubule plus-ends and finally lead to the formation of parallel bundles. At the same time, Kif15 motors cooperate to suppress catastrophe events at polymerizing microtubule plus-ends, raising the possibility that Kif15 motors may synchronize the dynamics of bundles that they have assembled. Thus, Kif15 is adapted to operate on parallel microtubule substrates, a property that clearly distinguishes it from the other tetrameric spindle motor, Eg5.

The Enterohemorrhagic Escherichia coli Effector EspW Triggers Actin Remodeling in a Rac1-Dependent Manner.

Enterohemorrhagic Escherichia coli (EHEC) is a diarrheagenic pathogen that colonizes the gut mucosa and induces attaching-and-effacing lesions. EHEC employs a type III secretion system (T3SS) to translocate 50 effector proteins that hijack and manipulate host cell signaling pathways, which allow bacterial colonization and subversion of immune responses and disease progression. The aim of this study was to characterize the T3SS effector EspW. We found espW in the sequenced O157:H7 and non-O157 EHEC strains as well as in Shigella boydii Furthermore, a truncated version of EspW, containing the first 206 residues, is present in EPEC strains belonging to serotype O55:H7. Screening a collection of clinical EPEC isolates revealed that espW is present in 52% of the tested strains. We report that EspW modulates actin dynamics in a Rac1-dependent manner. Ectopic expression of EspW results in formation of unique membrane protrusions. Infection of Swiss cells with an EHEC espW deletion mutant induces a cell shrinkage phenotype that could be rescued by Rac1 activation via expression of the bacterial guanine nucleotide exchange factor, EspT. Furthermore, using a yeast two-hybrid screen, we identified the motor protein Kif15 as a potential interacting partner of EspW. Kif15 and EspW colocalized in cotransfected cells, while ectopically expressed Kif15 localized to the actin pedestals following EHEC infection. The data suggest that Kif15 recruits EspW to the site of bacterial attachment, which in turn activates Rac1, resulting in modifications of the actin cytoskeleton that are essential to maintain cell shape during infection.

Yeast GSK-3 kinase regulates astral microtubule function through phosphorylation of the microtubule-stabilizing kinesin Kip2.

The S. cerevisiae kinesin Kip2 stabilises astral microtubules (MTs) and facilitates spindle positioning through transport of MT-associated proteins, such as the yeast CLIP-170 homologue Bik1, dynein and the adenomatous-polyposis-coli-related protein Kar9 to the plus ends of astral MTs. Here, we show that Kip2 associates with its processivity factor Bim1, the yeast homologue of the plus-end-tracking protein EB1. This interaction requires an EB1-binding motif in the N-terminal extension of Kip2 and is negatively regulated by phosphorylation through Mck1, the yeast glycogen synthase kinase 3. In addition, Mck1-dependent phosphorylation decreases the intrinsic MT affinity of Kip2. Reduction in Kip2 phosphorylation leads to stabilisation of astral MTs, and accumulation of Kip2, dynein and Kar9 at MT plus ends, whereas loss of Mck1 function leads to defects in spindle positioning. Furthermore, we provide evidence that a subpopulation of Mck1 at the bud-cortex phosphorylates Kip2. We propose that yeast GSK-3 spatially controls astral MT dynamics and the loading of dynein and Kar9 on astral MT plus ends by regulating Kip2 interactions with Bim1 and MTs.

Triggered contraction of self-assembled micron-scale DNA nanotube rings.

Contractile rings are formed from cytoskeletal filaments during cell division. Ring formation is induced by specific crosslinkers, while contraction is typically associated with motor protein activity. Here, we engineer DNA nanotubes and peptide-functionalized starPEG constructs as synthetic crosslinkers to mimic this process. The crosslinker induces bundling of ten to hundred DNA nanotubes into closed micron-scale rings in a one-pot self-assembly process yielding several thousand rings per microliter. Molecular dynamics simulations reproduce the detailed architectural properties of the DNA rings observed in electron microscopy. Theory and simulations predict DNA ring contraction - without motor proteins - providing mechanistic insights into the parameter space relevant for efficient nanotube sliding. In agreement between simulation and experiment, we obtain ring contraction to less than half of the initial ring diameter. DNA-based contractile rings hold promise for an artificial division machinery or contractile muscle-like materials.

CENP-V is required for proper chromosome segregation through interaction with spindle microtubules in mouse oocytes.

Proper chromosome segregation is essential to avoid aneuploidy, yet this process fails with increasing age in mammalian oocytes. Here we report a role for the scarcely described protein CENP-V in oocyte spindle formation and chromosome segregation. We show that depending on the oocyte maturation state, CENP-V localizes to centromeres, to microtubule organizing centers, and to spindle microtubules. We find that Cenp-V-/- oocytes feature severe deficiencies, including metaphase I arrest, strongly reduced polar body extrusion, increased numbers of mis-aligned chromosomes and aneuploidy, multipolar spindles, unfocused spindle poles and loss of kinetochore spindle fibres. We also show that CENP-V protein binds, diffuses along, and bundles microtubules in vitro. The spindle assembly checkpoint arrests about half of metaphase I Cenp-V-/- oocytes from young adults only. This finding suggests checkpoint weakening in ageing oocytes, which mature despite carrying mis-aligned chromosomes. Thus, CENP-V is a microtubule bundling protein crucial to faithful oocyte meiosis, and Cenp-V-/- oocytes reveal age-dependent weakening of the spindle assembly checkpoint.

Multivalent electrostatic microtubule interactions of synthetic peptides are sufficient to mimic advanced MAP-like behavior.

Microtubule-associated proteins (MAPs) are a functionally highly diverse class of proteins that help to adjust the shape and function of the microtubule cytoskeleton in space and time. For this purpose, MAPs structurally support microtubules, modulate their dynamic instability, or regulate the activity of associated molecular motors. The microtubule-binding domains of MAPs are structurally divergent, but often depend on electrostatic interactions with the negatively charged surface of the microtubule. This suggests that the surface exposure of positive charges rather than a certain structural fold is sufficient for a protein to associate with microtubules. Consistently, positively charged artificial objects have been shown to associate with microtubules and to diffuse along their lattice. Natural MAPs, however, show a more sophisticated functionality beyond lattice-diffusion. Here, we asked whether basic electrostatic interactions are sufficient to also support advanced MAP functionality. To test this hypothesis, we studied simple positively charged peptide sequences for the occurrence of typical MAP-like behavior. We found that a multivalent peptide construct featuring four lysine-alanine heptarepeats (starPEG-(KA7)4)-but not its monovalent KA7-subunits-show advanced, biologically relevant MAP-like behavior: starPEG-(KA7)4 binds microtubules in the low nanomolar range, diffuses along their lattice with the ability to switch between intersecting microtubules, and tracks depolymerizing microtubule ends. Further, starPEG-(KA7)4 promotes microtubule nucleation and growth, mediates depolymerization coupled pulling at plus ends, and bundles microtubules without significantly interfering with other proteins on the microtubule lattice (as exemplified by the motor kinesin-1). Our results show that positive charges and multivalency are sufficient to mimic advanced MAP-like behavior.

Kif15 functions as an active mechanical ratchet.

Kif15 is a kinesin-12 that contributes critically to bipolar spindle assembly in humans. Here we use force-ramp experiments in an optical trap to probe the mechanics of single Kif15 molecules under hindering or assisting loads and in a variety of nucleotide states. While unloaded Kif15 is established to be highly processive, we find that under hindering loads, Kif15 takes <∼10 steps. As hindering load is increased, Kif15 forestep:backstep ratio decreases exponentially, with stall occurring at 6 pN. In contrast, under assisting loads, Kif15 detaches readily and rapidly, even from its AMPPNP state. Kif15 mechanics thus depend markedly on the loading direction. Kif15 interacts with a binding partner, Tpx2, and we show that Tpx2 locks Kif15 to microtubules under both hindering and assisting loads. Overall, our data predict that Kif15 in the central spindle will act as a mechanical ratchet, supporting spindle extension but resisting spindle compression.

Interleukin 27 induces differentiation of neural C6-precursor cells into astrocytes.

Interleukin 6 (IL6)-type cytokines are major regulators of inflammation and thereby contribute to the neuropathology and pathophysiology associated with inflammation of the central nervous system (CNS). Furthermore, astrocyte development which is a key process in the development of the CNS is also controlled by cytokines of the IL6-family. Interleukin 27 (IL27) is a recently identified member of this family and has been implicated in the inhibition of TH17 T-cell-responses. Here we show that IL27 and the HHV8 encoded viral IL6 (vIL6) induce C6 glioma cells to differentiate into an astrocyte-like state. Cytokine stimulation led to STAT-factor phosphorylation and consequently to protein expression of the astrocyte marker glial fibrillary acidic protein (GFAP). These data could be confirmed by GFAP-immunostaining of stimulated cells. Taken together, IL27 and vIL6 can be considered as new astrocyte-inducing cytokines of the brain.

Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation.

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1's error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex's conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

The Kinesin-12 Kif15 is a processive track-switching tetramer.

Kinesin-12 motors are a little studied branch of the kinesin superfamily with the human protein (Kif15) implicated in spindle mechanics and chromosome movement. In this study, we reconstitute full-length hKif15 and its microtubule-targeting factor hTpx2 in vitro to gain insight into the motors mode of operation. We reveal that hKif15 is a plus-end-directed processive homotetramer that can step against loads of up to 3.5 pN. We further show that hKif15 is the first kinesin that effectively switches microtubule tracks at intersections, enabling it to navigate microtubule networks, such as the spindle. hKif15 tetramers are also capable of cross-linking microtubules, but unexpectedly, this does not depend on hTpx2. Instead, we find that hTpx2 inhibits hKif15 stepping when microtubule-bound. Our data reveal that hKif15 is a second tetrameric spindle motor in addition to the kinesin-5 Eg5 and provides insight into the mechanisms by which hKif15 and its inhibitor hTpx2 modulate spindle microtubule architecture. DOI: http://dx.doi.org/10.7554/eLife.01724.001.

Exotic mitotic mechanisms.

The emergence of eukaryotes around two billion years ago provided new challenges for the chromosome segregation machineries: the physical separation of multiple large and linear chromosomes from the microtubule-organizing centres by the nuclear envelope. In this review, we set out the diverse solutions that eukaryotic cells use to solve this problem, and show how stepping away from 'mainstream' mitosis can teach us much about the mechanisms and mechanics that can drive chromosome segregation. We discuss the evidence for a close functional and physical relationship between membranes, nuclear pores and kinetochores in generating the forces necessary for chromosome segregation during mitosis.