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Posttranslational Modifications and Activity of Purified Tubulin (A) Western blot against tubulin PTMs before and after the purification. 300 ng tubulin for each species (Bos taurus, Xenopus laevis, Xenopus tropicalis) were loaded onto a polyacrylamide gel adjacent to a corresponding volume of egg extract. Samples were transferred to a nitrocellulose membrane and probed with antibodies against a-tubulin, tyrosinated tubulin (Tyr), detyrosinated tubulin (Detyr), acetylated lysine 40 (K40), and phosphoserine (Pser). (B) Tubulin polymerization assay for assessing the relative activities of bovine brain tubulin and Xenopus tubulin purified via the TOG column. 2 mL of the 20 mM initial reaction mixture was loaded onto the gel as the input (I), followed by equivalent volumes for the supernatant (S) and the pellet (P) after resuspending the pellet in the same starting volume of 13 BRB80.
Source publication
Cytoplasmic extracts from unfertilized Xenopus eggs have made important contributions to our understanding of microtubule dynamics, spindle assembly, and scaling. Until recently, these in vitro studies relied on the use of heterologous tubulin. This protocol allows for the purification of physiologically relevant Xenopus tubulins in milligram yield...
Contexts in source publication
Context 1
... make sure that the purification did not change the tubulin's post-translational modification pattern, we probe extract tubulin (sample taken at step 26) and purified tubulin (sample taken at step 56) by western blot using antibodies against a-tubulin as loading control, tyrosinated tubulin (Tyr), detyrosinated tubulin (Detyr), acetylated lysine 40 (K40), and phosphoserine (P-Ser) ( Figure 5A). ...
Citations
... Metaphase-arrested egg extract was prepared from laid X. laevis eggs as previously described (106,107). Briefly, X. laevis frogs were primed with 100 U of pregnant mare serum gonadotrophin (PMSG) 3-7 days before the experiment and were boosted with 1000 U human chorionic gonadotrophin (HCG) to induce egg laying. ...
The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.
... Purification of GST-TOG1/2 followed previously described methods and is described briefly below (Reusch et al., 2020;Widlund et al., 2012). The pGEX-6P-1 Stu2 1-590 plasmid was transformed into BL21 cells and colonies were grown on an LB plate containing 100 µg/ml carbenicillin and 15 µg/ ml chloramphenicol at 37°C. ...
Heterozygous, missense mutations in a- or b-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether a mutation’s impact at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A -V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and a decrease in the number of neurite retraction events in primary rat neuronal cultures. These neuronal phenotypes are accompanied by increased microtubule acetylation and polymerization rates. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends in budding yeast and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A -V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A -V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations disrupt microtubule regulation typically conferred by XMAP215 proteins during neuronal morphogenesis and migration, and this impact on tubulin activity at the molecular level scales with the impact at the cellular and tissue levels.
... Another coverslip was immediately placed on this coverslip to create a squashed egg extract sample, which provided a thin layer that could be easily imaged with our imaging setup. For the tubulin addition assays, assembly-competent X. laevis tubulin was purified using a TOG-column as in (Widlund et al., 2012;Reusch et al., 2020). In brief, the extract was first diluted with an equal volume of BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl 2 , pH 6.9) and centrifuged at 80,000 rpm in an MLA-80 rotor (Beckman-Coulter) for 10 min at 4 C. ...
... For other tubulin sources, this protocol might require empirical optimization. For example, room temperature is sufficient to allow growth of microtubule seeds using Xenopus laevis tubulin Reusch et al., 2020). ...
Dynamic microtubules are essential for many processes in the lives of eukaryotic cells. To study and understand the mechanisms of microtubule dynamics and regulation, in vitro reconstitution with purified components has proven a vital approach. Imaging microtubule dynamics can be instructive for a given species, isoform composition, or biochemical modification. Here, we describe two methods that visualize microtubule dynamics at high speed and high contrast: (1) total internal reflection fluorescence microscopy and (2) label-free interference reflection microscopy.
For complete details on the use and execution of this protocol, please refer to Hirst et al. (2020).
Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets.1, 2, 3, 4, 5 While microtubules and their molecular building block αβ-tubulin are established drug targets in a variety of cancers,⁶,⁷ we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and β-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials.
Heterozygous, missense mutations in α- or β-tubulin genes are associated with a wide range of human brain malformations, known as tubulinopathies. We seek to understand whether the impact of a mutation at the molecular and cellular levels scale with the severity of brain malformation. Here we focus on two mutations at the valine 409 residue of TUBA1A, V409I and V409A, identified in patients with pachygyria or lissencephaly, respectively. We find that ectopic expression of TUBA1A-V409I/A mutants disrupt neuronal migration in mice and promote excessive neurite branching and delayed retraction in primary neuronal cultures, accompanied by increased microtubule acetylation. To determine the molecular mechanisms, we modeled the V409I/A mutants in budding yeast and found that they promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin. In addition, V409I/A mutants decrease the recruitment of XMAP215/Stu2 to plus ends and ablate tubulin binding to TOG domains. In each assay tested, the TUBA1A-V409I mutant exhibits an intermediate phenotype between wild type and the more severe TUBA1A-V409A, reflecting the severity observed in brain malformations. Together, our data support a model in which the V409I/A mutations may limit tubulin conformational states and thereby disrupt microtubule regulation during neuronal morphogenesis and migration.
The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle’s material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle’s mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle’s emergent physical properties—essential to advance predictive frameworks of spindle assembly and function.
