Arshad Desai

Kin I Kinesins Are Microtubule-Destabilizing Enzymes

Using in vitro assays with purified proteins, we show that XKCM1 and XKIF2, two distinct members of the internal catalytic domain (Kin I) kinesin subfamily, catalytically destabilize microtubules using a novel mechanism. Both XKCM1 and XKIF2 influence microtubule stability by targeting directly to microtubule ends where they induce a destabilizing conformational change. ATP hydrolysis recycles XKCM1/XKIF2 for multiple rounds of action by dissociating a XKCM1/ XKIF2-tubulin dimer complex released upon microtubule depolymerization. These results establish Kin I kinesins as microtubule-destabilizing enzymes, distinguish them mechanistically from kinesin superfamily members that use ATP hydrolysis to translocate along microtubules, and have important implications for the regulation of microtubule dynamics and for the intracellular functions and evolution of the kinesin superfamily.

XKCM1: A Xenopus Kinesin-Related Protein That Regulates Microtubule Dynamics during Mitotic Spindle Assembly

We isolated a cDNA clone encoding a kinesin-related protein, which we named XKCM1. Antibodies to XKCM1 stain mitotic centromeres and spindle poles. Immunodepletion and antibody addition experiments in an in vitro spindle assembly assay show that XKCM1 is required for both establishment and maintenance of mitotic spindles. The structures that form in the absence of XKCM1 contain abnormally long microtubules. This long microtubule defect can be rescued by the addition of purified XKCM1 protein. Analysis of microtubule dynamics in a clarified mitotic extract reveals that loss of XKCM1 function causes a 4-fold suppression in the catastrophe frequency. XKCM1 thus exhibits a novel activity for a kinesin-related protein by promoting microtubule depolymerization during mitotic spindle assembly.

The spindle: a dynamic assembly of microtubules and motors

In all eukaryotes, a microtubule-based structure known as the spindle is responsible for accurate chromosome segregation during cell division. Spindle assembly and function require localized regulation of microtubule dynamics and the activity of a variety of microtubule-based motor proteins. Recent work has begun to uncover the molecular mechanisms that underpin this process. Here we describe the structural and dynamic properties of the spindle, and introduce the current concepts regarding how a bipolar spindle is assembled and how it functions to segregate chromosomes.

Fluorescent speckle microscopy, a method to visualize the dynamics of protein assemblies in living cells

Fluorescence microscopic visualization of fluorophore-conjugated proteins that have been microinjected or expressed in living cells and have incorporated into cellular structures has yielded much information about protein localization and dynamics [1]. This approach has, however, been limited by high background fluorescence and the difficulty of detecting movement of fluorescent structures because of uniform labeling. These problems have been partially alleviated by the use of more cumbersome methods such as three-dimensional confocal microscopy, laser photobleaching and photoactivation of fluorescence [2]. We report here a method called fluorescent speckle microscopy (FSM) that uses a very low concentration of fluorescent subunits, conventional wide-field fluorescence light microscopy and digital imaging with a low-noise, cooled charged coupled device (CCD) camera. A unique feature of this method is that it reveals the assembly dynamics, movement and turnover of protein assemblies throughout the image field of view at diffraction-limited resolution. We found that FSM also significantly reduces out-of-focus fluorescence and greatly improves visibility of fluorescently labeled structures and their dynamics in thick regions of living cells. Our initial applications include the measurement of microtubule movements in mitotic spindles and actin retrograde flow in migrating cells.

The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.

Publisher Summary This chapter presents detailed methods for the preparation of cytostatic factor (CSF) extracts and for performing spindle assembly reactions. It also describes methods for depleting specific components from extracts, an approach that has been used successfully to determine the contributions of both motor and nonmotor components to spindle assembly. It also describes methods for analyzing anaphase in vitro. Mature Xenopus eggs are arrested in metaphase of meiosis II by CSF, which is thought to be the product of the c-mos protooncogene. Sperm entry triggers a calcium spike that initiates a series of events leading to the destruction of CSF and exit from the meiosis II metaphase arrest. This calcium sensitivity of the CSF arrest is exploited in the preparation of extracts by use of the calcium chelator EGTA. The presence of EGTA in buffers results in extracts that maintain the CSF arrest but can be induced to exit the CSF arrest by addition of calcium. This convenient control of cell cycle state allows one to easily obtain in vitro spindles with replicated chromosomes.

FLUORESCENT SPECKLE MICROSCOPY OF SPINDLE MICROTUBULE ASSEMBLY AND MOTILITY IN LIVING CELLS

Publisher Summary This chapter introduces the principles of the fluorescent speckle microscopy method for microtubules, and then briefly describes the features of a digital imaging system that are important for speckle image recording and analysis with other optical modes, such as phase contrast or vital staining, with DNA dyes such as the blue fluorescent Hoescht or DAPI to record chromosome position. Applications of the method are presented for measuring microtubule movements in tissue cell spindles and asters and in spindles reassembled in vitro in cytoplasmic extracts of Xenopus eggs. A high-resolution, multimode, multiwavelength, digital fluorescence light microscope is used for fluorescence imaging of mitosis in living cells or cytoplasmic extracts that have enough sensitivity for fluorescence speckle imaging. Images of chromosomes and cell structure can also be obtained by transmitted light differential interference contrast (DIC) or phase contrast. The microscope utilizes conventional wide field optics and a cooled, slow-scan CCD camera for image detection.

Tubulin and FtsZ structures: functional and therapeutic implications

The microtubule cytoskeleton has lagged nearly a decade behind the actin cytoskeleton with respect to structural information on the basic polymer subunit. This structural inferiority complex has finally been lifted by two recent papers describing the structures of the alpha beta tubulin dimer and FtsZ, a protein similar to tubulin that is essential for cell division in prokaryotes.