Single molecule mechanics of Kif15

Kinesin-12 is a motor protein that has a role in the processes of mitotic spindle formation and maintenance. The human Kinesin-12, Kif15, has been shown to have some functional redundancy with Eg5, a Kinesin-5 that plays key roles in the formation of the bipolar spindle and is a potential target for anti-cancer drugs. Eg5 is thought to contribute to spindle formation by cross-linking and sliding microtubules, however little is known about the mechanism of Kif15. We have used laser tweezers to investigate the mechanical properties of Kif15 compared to those of kinesin-1. We have found that Kif15 is plus end directed and takes multiple steps along the microtubule without detaching. Full-length Kif15 walks faster and supports more load than full-length Eg5. Kif15 is less processive under load than kinesin-1, although it has a similar stall force. A second, diffusive, microtubule binding site in Kif15 supports processivity at zero load, and slows flyback following a detachment in the optical trap. The microtubule-associated protein, Tpx2, is necessary for the localisation of Kif15 to spindle microtubules. We find that Tpx2 binding arrests the motion of Kif15 and creates a stable binding state that resists both assisting and hindering loads. We also find evidence of a tail-mediated auto-inhibitory mechanism that creates a stable MT binding state and causes pausing during processive runs. C-terminal truncation of the Kif15 tail relieves this inhibition leading to faster overall stepping and abrogates the effects of Tpx2. We examined the detachment behaviour of Kif15 from microtubules, under assisting and hindering loads. We find that assisting loads cause single Kif15 and Kinesin-1 motors to detach from the microtubule more easily than hindering loads. Kif15 shows a much more asymmetric response to load in low levels of ATP than Kinesin-1, and both show more asymmetry than Eg5: previous work has shown that the behaviour of Eg5 does not change dramatically with differing loading directions. This has interesting implications for the roles of Kif15 and Eg5 motors in both parallel and anti-parallel microtubule bundles. Overall our data supports an in vivo mechanism for Kif15 that it distinct from that of Eg5. We investigated the load-dependent detachment of Kinesin-1 and Kif15 in millimolar concentrations of ADP, AMPPNP and micromolar concentrations of ATP. Kinesin-1 in ADP detached at low loads, and in AMPPNP at two different loads, both higher than in ADP. These two AMPPNP states of Kinesin1 likely corresponding to single and double headed microtubule binding, as proposed by Ishiwata and colleagues. Kif15 behaved broadly similarly. At micromolar ATP concentrations and hindering loads, both Kinesin-1 and Kif15 again showed two different high load detachment states. This is inconsistent with the model proposed by Ishiwata and possible modifications are discussed

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. Whilst 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. By 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

Establishment of centromere identity is dependent on nuclear spatial organization

The establishment of centromere-specific CENP-A chromatin is influenced by epigenetic and genetic processes. Central domain sequences from fission yeast centromeres are preferred substrates for CENP-A(Cnp1) incorporation, but their use is context dependent, requiring adjacent heterochromatin. CENP-A(Cnp1) overexpression bypasses heterochromatin dependency, suggesting that heterochromatin ensures exposure to conditions or locations permissive for CENP-A(Cnp1) assembly. Centromeres cluster around spindle-pole bodies (SPBs). We show that heterochromatin-bearing minichromosomes localize close to SPBs, consistent with this location promoting CENP-A(Cnp1) incorporation. We demonstrate that heterochromatin-independent de novo CENP-A(Cnp1) chromatin assembly occurs when central domain DNA is placed near, but not far from, endogenous centromeres or neocentromeres. Moreover, direct tethering of central domain DNA at SPBs permits CENP-A(Cnp1) assembly, suggesting that the nuclear compartment surrounding SPBs is permissive for CENP-A(Cnp1) incorporation because target sequences are exposed to high levels of CENP-A(Cnp1) and associated assembly factors. Thus, nuclear spatial organization is a key epigenetic factor that influences centromere identity

Young adult born neurons enhance hippocampal dependent performance via influences on bilateral networks

Adult neurogenesis supports performance in many hippocampal dependent tasks. Considering the small number of adult-born neurons generated at any given time, it is surprising that this sparse population of cells can substantially influence behavior. Recent studies have demonstrated that heightened excitability and plasticity may be critical for the contribution of young adult-born cells for certain tasks. What is not well understood is how these unique biophysical and synaptic properties may translate to networks that support behavioral function. Here we employed a location discrimination task in mice while using optogenetics to transiently silence adult-born neurons at different ages. We discovered that adult-born neurons promote location discrimination during early stages of development but only if they undergo maturation during task acquisition. Silencing of young adult-born neurons also produced changes extending to the contralateral hippocampus, detectable by both electrophysiology and fMRI measurements, suggesting young neurons may modulate location discrimination through influences on bilateral hippocampal networks.United States. National Institutes of Health (1DP2NS082126)National Institute of Mental Health (U.S.) (5R00MH085944)United States. National Institutes of Health (R01-DA028299)United States. Defense Advanced Research Projects Agency (W911NF-10-0059)Pew Charitable TrustsAmerican Federation for Aging ResearchAlfred P. Sloan FoundationNational Institute of Mental Health (U.S.) (1R21MH109941

The Witan, 1980-1981 Academic Year V. 9 No. 2, October 1980

SBA Appropriates Funds, Twenty Eighth Red Mass, Chili Team Loses San Marcos Meet, Guinea Pig Treatment for Cunningham, Dean Castleberry's Accreditation Efforts Lauded, The President's Report Graduation Fee Increase, Intermediate Criminal Court Jurisdicti

The Witan, 1980-1981 Academic Year V. 9 No. 2, October 1980

SBA Appropriates Funds, Twenty Eighth Red Mass, Chili Team Loses San Marcos Meet, Guinea Pig Treatment for Cunningham, Dean Castleberry's Accreditation Efforts Lauded, The President's Report Graduation Fee Increase, Intermediate Criminal Court Jurisdicti