One of the most intriguing aspects of mitosis is the ability of kinetochores to hold onto plus ends of microtubules that are actively gaining or losing tubulin subunits. Here, we show that CLASP1, a microtubule-associated protein, localizes preferentially near the plus ends of growing spindle microtubules and is also a component of a kinetochore region that we term the outer corona. A truncated form of CLASP1 lacking the kinetochore binding domain behaves as a dominant negative, leading to the formation of radial arrays of microtubule bundles that are highly resistant to depolymerization. Microinjection of CLASP1-specific antibodies suppresses microtubule dynamics at kinetochores and throughout the spindle, resulting in the formation of monopolar asters with chromosomes buried in the interior. Incubation with microtubule-stabilizing drugs rescues the kinetochore association with microtubule plus ends at the periphery of the asters. Our data suggest that CLASP1 is required at kinetochores for attached microtubules to exhibit normal dynamic behavior.

A favored hypothesis to explain the pathology underlying nuclear envelopathies is that mutations in nuclear envelope proteins alter genome/chromatin organization and thus gene expression. To identify nuclear envelope proteins that play roles in genome organization, we analyzed nuclear envelopes from resting and phytohemagglutinin-activated leukocytes because leukocytes have a particularly high density of peripheral chromatin that undergoes significant reorganization upon such activation. Thus, nuclear envelopes were isolated from leukocytes in the two states and analyzed by multidimensional protein identification technology using an approach that used expected contaminating membranes as subtractive fractions. A total of 3351 proteins were identified between both nuclear envelope data sets among which were 87 putative nuclear envelope transmembrane proteins (NETs) that were not identified in a previous proteomics analysis of liver nuclear envelopes. Nuclear envelope localization was confirmed for 11 new NETs using tagged fusion proteins and antibodies on spleen cryosections. 27% of the new proteins identified were unique to one or the other of the two leukocyte states. Differences in expression between activated and resting leukocytes were confirmed for some NETs by RT-PCR, and most of these proteins appear to only be expressed in certain types of blood cells. Several known proteins identified in both data sets have functions in chromatin organization and gene regulation. To test whether the novel NETs identified might include those that also regulate chromatin, nine were run through two screens for different chromatin effects. One screen found two NETs that can recruit a specific gene locus to the nuclear periphery, and the second found a different NET that promotes chromatin condensation. The variation in the protein milieu with pharmacological activation of the same cell population and consequences for gene regulation suggest that the nuclear envelope is a complex regulatory system with significant influences on genome organization.

Running title: Chemical extraction of nuclear membrane proteins 2 Key Words: nuclear envelope (NE); endoplasmic reticulum (ER); microsomal membrane (MM); integral membrane protein; inner nuclear membrane (INM); outer nuclear membrane (ONM); detergent; chaotrope; alkaline; multi-dimensional protein identification technology (MudPIT). 3 AbstractThe nuclear envelope (NE) is a double membrane system that is both a part of the endoplasmic reticulum and part of the nucleus. As its constituent proteins tend to be highly complexed with nuclear and cytoplasmic components, it is notoriously difficult to purify. Two methods can reduce this difficulty for the identification of nuclear membrane proteins: comparison to contaminating membranes and chemical extractions to enrich for certain groups of proteins. The purification of nuclear envelopes and contaminating microsomal membranes is described here along with procedures for chemical extraction using salt and detergent, chaotropes, or alkaline solutions. Each extraction method enriches for different combinations of nuclear envelope proteins. Finally, we describe the analysis of these fractions with MudPIT, a proteomics methodology that avoids gel extraction of bands to facilitate identification of minor proteins and membrane proteins that do not resolve well on gels. Together these three approaches can significantly increase the output of proteomics studies aimed at identifying the protein complement of subcellular membrane systems.4