Lynne Cassimeris

The oncoprotein 18/stathmin family of microtubule destabilizers

The past several years have seen major advances in our understanding of the mechanisms of microtubule destabilization by oncoprotein18/stathmin (Op18/stathmin) and related proteins. New structural information has clearly shown how members of the Op18/stathmin protein family bind tubulin dimers and suggests models for how these proteins stimulate catastrophe, the transition from microtubule growth to shortening. Regulation of Op18/stathmin by phosphorylation continues to capture much attention. Studies suggest that phosphorylation occurs in a localized fashion, resulting in decreased microtubule destabilizing activity near chromatin or microtubule polymer. A spatial gradient of inactive Op18/stathmin associated with chromatin or microtubules could contribute significantly to mitotic spindle assembly.

Accessory protein regulation of microtubule dynamics throughout the cell cycle

A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle.

Regulation of microtubule-associated proteins

Microtubule-associated proteins (MAPs) function to regulate the assembly dynamics and organization of microtubule polymers. Upstream regulation of MAP activities is the major mechanism used by cells to modify and control microtubule assembly and organization. This review summarizes the functional activities of MAPs found in animal cells and discusses how these MAPs are regulated. Mechanisms controlling gene expression, isoform-specific expression, protein localization, phosphorylation, and degradation are discussed. Additional regulatory mechanisms include synergy or competition between MAPs and the activities of cofactors or binding partners. For each MAP it is likely that regulation in vivo reflects a composite of multiple regulatory mechanisms.

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.

Estimates of lateral and longitudinal bond energies within the microtubule lattice

We developed a stochastic model of microtubule (MT) assembly dynamics that estimates tubulin–tubulin bond energies, mechanical energy stored in the lattice dimers, and the size of the tubulin-GTP cap at MT tips. First, a simple assembly/disassembly state model was used to screen possible combinations of lateral bond energy (ΔGLat) and longitudinal bond energy (ΔGLong) plus the free energy of immobilizing a dimer in the MT lattice (ΔGS) for rates of MT growth and shortening measured experimentally. This analysis predicts ΔGLat in the range of −3.2 to −5.7 kBT and ΔGLong plus ΔGS in the range of −6.8 to −9.4 kBT. Based on these estimates, the energy of conformational stress for a single tubulin-GDP dimer in the lattice is 2.1–2.5 kBT. Second, we studied how tubulin-GTP cap size fluctuates with different hydrolysis rules and show that a mechanism of directly coupling subunit addition to hydrolysis fails to support MT growth, whereas a finite hydrolysis rate allows growth. By adding rules to mimic the mechanical constraints present at the MT tip, the model generates tubulin-GTP caps similar in size to experimental estimates. Finally, by combining assembly/disassembly and cap dynamics, we generate MT dynamic instability with rates and transition frequencies similar to those measured experimentally. Our model serves as a platform to examine GTP-cap dynamics and allows predictions of how MT-associated proteins and other effectors alter the energetics of MT assembly.

The Interaction of TOGp with Microtubules and Tubulin

TOGp is the human homolog of XMAP215, aXenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.

Phosphorylation by CDK1 regulates XMAP215 function in vitro.

XMAP215, a microtubule-associated protein isolated from Xenopus eggs, promotes microtubule assembly dynamics in an end-specific manner: addition of XMAP215 to purified porcine tubulin increases both elongation and shortening rates at microtubule plus ends, with minimal effects at minus ends. Previous results indicated that XMAP215 is phosphorylated during M phase, suggesting that its activity may be regulated by cell cycle phosphorylation. To test this hypothesis, we used video-enhanced DIC microscopy to examine the effects of XMAP215 phosphorylated by CDK1 on the assembly of purified tubulin. XMAP215 incubated with ATP at 30 degrees C for 10- 20 min in the absence of CDK1 exhibited a 4.1-fold increase in plus end elongation rate compared to purified tubulin. Elongation was promoted to a lesser degree (2.4-fold) by phosphorylated XMAP215. In contrast, XMAP215 phosphorylation did not alter the approximately 3-fold increase in shortening rate. XMAP215 binding to taxol microtubules was also not changed by phosphorylation. To further investigate mechanisms responsible for the faster microtubule shortening rate observed with XMAP215, we made microtubules with segments assembled prior to XMAP215 addition (proximal segments) and segments assembled in the presence of XMAP215 (distal segments). In 9 of 10 microtubules, the distal segment shortened faster (distal = 60.7 microm/min; proximal = 37.5 microm/min), suggesting that MTs assembled in the presence of XMAP215 have an altered lattice that results in subsequent faster shortening.

In vitro assembled plant microtubules exhibit a high state of dynamic instability.

Higher plants possess four distinct microtubule arrays. One of these, the cortical array, is involved in orienting the deposition of cellulose microfibrils. This plant interphase array is also notable because it contains exceptionally dynamic microtubules. Although the primary sequence of plant and animal tubulin is similar (79-87% amino acid identity overall) there are some regions of divergence. Thus, one possible explanation for the high state of polymer assembly and turnover that is observed in plant interphase arrays is that the tubulins have evolved differently and possess a higher intrinsic dynamic character than their animal counterparts. This hypothesis was tested using highly purified plant tubulin assembled in vitro. Using high-resolution DIC video-enhanced microscopy, we quantified the four characteristic parameters of dynamic instability of plant microtubules and compared them with animal microtubules. The elongation velocities between plant and animal microtubules are similar, but plant microtubules undergo catastrophes more frequently, do not exhibit any rescues, and have an average shortening velocity of 195 microm/min (compared with 21 microm/min for animal microtubules). These data support the hypothesis that plant tubulin forms microtubules that are intrinsically more dynamic than those of animals.

Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E

Factors that regulate the microtubule cytoskeleton are critical in determining cell behavior. Here we describe the function of a novel protein that we term E-like based on its sequence similarity to the tubulin-specific chaperone cofactor E. We find that upon overexpression, E-like depolymerizes microtubules by committing tubulin to proteosomal degradation. Our data suggest that this function is direct and is based on the ability of E-like to disrupt the tubulin heterodimer in vitro. Suppression of E-like expression results in an increase in the number of stable microtubules and a tight clustering of endocellular membranes around the microtubule-organizing center, while the properties of dynamic microtubules are unaffected. These observations define E-like as a novel regulator of tubulin stability, and provide a link between tubulin turnover and vesicle transport.

Stathmin Regulates Centrosomal Nucleation of Microtubules and Tubulin Dimer/Polymer Partitioning

Stathmin is a microtubule-destabilizing protein ubiquitously expressed in vertebrates and highly expressed in many cancers. In several cell types, stathmin regulates the partitioning of tubulin between unassembled and polymer forms, but the mechanism responsible for partitioning has not been determined. We examined stathmin function in two cell systems: mouse embryonic fibroblasts (MEFs) isolated from embryos +/+, +/-, and -/- for the stathmin gene and porcine kidney epithelial (LLCPK) cells expressing stathmin-cyan fluorescent protein (CFP) or injected with stathmin protein. In MEFs, the relative amount of stathmin corresponded to genotype, where cells heterozygous for stathmin expressed half as much stathmin mRNA and protein as wild-type cells. Reduction or loss of stathmin resulted in increased microtubule polymer but little change to microtubule dynamics at the cell periphery. Increased stathmin level in LLCPK cells, sufficient to reduce microtubule density, but allowing microtubules to remain at the cell periphery, also did not have a major impact on microtubule dynamics. In contrast, stathmin level had a significant effect on microtubule nucleation rate from centrosomes, where lower stathmin levels increased nucleation and higher stathmin levels reduced nucleation. The stathmin-dependent regulation of nucleation is only active in interphase; overexpression of stathmin-CFP did not impact metaphase microtubule nucleation rate in LLCPK cells and the number of astral microtubules was similar in stathmin +/+ and -/- MEFs. These data support a model in which stathmin functions in interphase to control the partitioning of tubulins between dimer and polymer pools by setting the number of microtubules per cell.