The minus-end depolymerase KIF2A drives flux-like treadmilling of γTuRC-uncapped microtubules.

During mitosis, microtubules in the spindle turn over continuously. At spindle poles, where microtubule minus ends are concentrated, microtubule nucleation and depolymerization, the latter required for poleward microtubule flux, happen side by side. How these seemingly antagonistic processes of nucleation and depolymerization are coordinated is not understood. Here, we reconstitute this coordination in vitro combining different pole-localized activities. We find that the spindle pole-localized kinesin-13 KIF2A is a microtubule minus-end depolymerase, in contrast to its paralog MCAK. Due to its asymmetric activity, KIF2A still allows microtubule nucleation from the γ-tubulin ring complex (γTuRC), which serves as a protective cap shielding the minus end against KIF2A binding. Efficient γTuRC uncapping requires the combined action of KIF2A and a microtubule severing enzyme, leading to treadmilling of the uncapped microtubule driven by KIF2A. Together, these results provide insight into the molecular mechanisms by which a minimal protein module coordinates microtubule nucleation and depolymerization at spindle poles consistent with their role in poleward microtubule flux

Determinants of polar versus nematic organization in networks of dynamic microtubules and mitotic motors

During cell division, mitotic motors organize microtubules in the bipolar spindle into either polar arrays at the spindle poles or a "nematic" network of aligned microtubules at the spindle center. The reasons for the distinct self-organizing capacities of dynamic microtubules and different motors are not understood. Using in vitro reconstitution experiments and computer simulations, we show that the human mitotic motors kinesin-5 KIF11 and kinesin-14 HSET, despite opposite directionalities, can both organize dynamic microtubules into either polar or nematic networks. We show that in addition to the motor properties the natural asymmetry between microtubule plus- and minus-end growth critically contributes to the organizational potential of the motors. We identify two control parameters that capture system composition and kinetic properties and predict the outcome of microtubule network organization. These results elucidate a fundamental design principle of spindle bipolarity and establish general rules for active filament network organization

The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability.

Microtubules are cytoskeletal polymers whose function depends on their property to switch between states of growth and shrinkage. Growing microtubules are thought to be stabilized by a GTP cap at their ends. The nature of this cap, however, is still poorly understood. End Binding proteins (EBs) recruit a diverse range of regulators of microtubule function to growing microtubule ends. Whether the EB binding region is identical to the GTP cap is unclear. Using mutated human tubulin with blocked GTP hydrolysis, we demonstrate that EBs bind with high affinity to the GTP conformation of microtubules. Slowing-down GTP hydrolysis leads to extended GTP caps. We find that cap length determines microtubule stability and that the microtubule conformation changes gradually in the cap as GTP is hydrolyzed. These results demonstrate the critical importance of the kinetics of GTP hydrolysis for microtubule stability and establish that the GTP cap coincides with the EB-binding region

Novel Kidins220/ARMS splice isoforms: potential specific regulators of neuronal and cardiovascular development

Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development

Expression of alternative splice isoforms of Kidins220 in adult mouse and human tissues.

<p>(A-B) RT-PCR analyses for exons encoding the amino-terminus of Kidins220 and between exons 24 and 30 (24f-30r) were carried out on adult mouse (A) and human (B) tissue panels. Tissues are labelled by capital letters. N indicates PCR products obtained using primers designed to recognise exons 3 and 8 in mouse, and exon 9 and 13 in human. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30. Arrowheads point to samples in which a specific alternative splicing pattern was detected. (C-D) Schematics of Kidins220 splice isoforms identified in mouse heart and brain (C) and human brain (D) in the region encoded by exons 24 to 30.</p

Identifying Juvenile Queen Conch Nurseries

Poster created by students of The Island School semester research class of Spring 201

Kidins220 alternative terminal exons in mouse and human tissue.

<p>(A-B) RT-PCR analysis for the carboxy-terminal endings of Kidins220 (exon 32 and exon 33) was carried out on an adult mouse (A) and human (B) tissue panels. Capital letters indicate the different tissues. 31f and 33 rev indicate amplification with primers against exons 31 and 33. Arrowheads point out samples where alternative terminal exon (ATE) splicing for Kidins220 is detected. (C) PCR products obtained using primers designed to recognise exons encoding the carboxy-terminus of Kidins220 (exons 31/32 and 32 in mouse and exon 31 and 32 in human).</p

Assessing Important Invertebrate Populations Within the Proposed Marine Protected Area in South Eleuthera

Poster created by students of The Island School semester research class of Spring 201

Distinct Kidins220 splice isoforms display specific cellular localisations.

<p>(A) Schematics of Kidins220 splice isoform m6 and Kidins220 ATE m6/C2 (from Exon 24 onwards) used for transfection of PC12 cells in Fig 7B. (B) PC12 cells were transfected with HA-tagged Kidins220 isoform m6, isoform m6/C2 or with Tet-ON pLVX vector only (control) and after 6 h stimulated with doxycycline and differentiated for 48 h with NGF. Full-length Kidins220 was detected using a polyclonal antibody directed against the carboxy-terminus of Kidins220 (GSC16 antibody; in green). An anti-HA antibody was used to stain Kidins220 isoforms m6 and m6/C2 (in red). The gain of the red channel was enhanced equally for cells overexpressing isoform m6/C2 and control cells, whilst it was tuned down for PC12 cells transfected with isoform m6 to adjust for the higher expression levels of this Kidins220 variant. Boxed areas of the merged images are magnified on the right. Representative pictures were chosen from three different experiments. Scale bars, 10 μm.</p

Developmental-specific expression of Kidins220 alternative splice isoforms in mouse brain.

<p>(A-B) RT-PCR analyses for exons encoding the amino-terminus of Kidins220 and between exons 24 and 30 (A), and for alternative terminal exon splicing (B) were carried out on mouse brains at different developmental stages, starting from embryonic stage E13.5 up to postnatal stage P64. The analysis at the latter stage includes adult male (M) and female (F) brains. N indicates PCR products obtained using primers designed to recognise exons 3 and 8. 24f-30r indicates samples obtained by amplification with primers recognising exons 24 and 30 (A). C indicates PCR products obtained using primers designed to recognise exons 31/32 and 32. 31f-33r indicates samples obtained by amplification with primers recognising exons 31 and 33 (B). The arrowhead points to appearance of Kidins220 isoform m1 (full-length) at postnatal stage P11 (A).</p