水蕨精子发生过程中中心体蛋白和微管蛋白的免疫荧光定位

采用透射电镜技术和免疫荧光标记技术对水蕨精子发生的超微结构以及中心体蛋白和微管蛋白在精子发生过程中的动态表达进行了观察。研究发现：（1）生毛体分化早期周围有放射状微管分布,这与线粒体向生毛体的聚集有关。（2）免疫荧光观察表明,中心体蛋白仅定位于生毛体、基体和鞭毛带上,自生毛体至基体阶段呈现明亮的荧光标记,在核塑形、鞭毛形成至精子成熟阶段,中心体蛋白荧光标记随着鞭毛的发生而逐渐减弱,至游动精子阶段中心体蛋白荧光标记信号几乎消失。（3）微管蛋白早期荧光标记与中心体蛋白标记形相同,在生毛体、鞭毛带、基体等运动细胞器上呈现明亮荧光标记,但微管蛋白随着鞭毛的发生其荧光标记越来越强。从二者的时空表达特征可以推断,中心体蛋白主要是运动细胞器的组织者,而非这些运动细胞器的结构成分,其功能是参与或负责中心粒、基体和鞭毛的发生。     

Sperm axoneme: a tale of tubulin posttranslation diversity

Two microtubule-containing structures are assembled during spermiogenesis: a transient manchette and a stable axoneme. Both structures contain microtubules enriched in posttranslationally modified tubulins. Despite the existence of a spectrum of tubulin isotypes postulated by the multi-tubulin hypothesis, further extended by an elaborated array of posttranslational modifications, it is unknown how this diversity influences microtubule function. There is increasing evidence that different alpha beta-tubulin isotypes can affect the structure and function of microtubules. It is also becoming increasingly clear that eukaryotic cells encode other tubulin proteins expressed by the tubulin superfamily: gamma, delta epsilon, zeta eta, and FtsZ have been identified so far. Although the role of gamma-tubulin in the nucleation of microtubule assembly is well established, the function of delta-, epsilon-, zeta-, eta-, and FtsZ-tubulins is less understood. The members of the tubulin superfamilies found in spermatids include the alpha beta-tubulin dimer, in addition to gamma-tubulin in the centrosome, and delta-tubulin in the perinuclear ring region of the mouse spermatid manchette, the centrosome region, and flagellum. Posttranslational modifications in tubulin isotypes are predominant in the C-terminus exposed on the outside surface of the microtubule. This target site may influence the interaction of microtubule-associated proteins, including motor proteins, and therefore determine the functional specificity of tubulin isotypes. It remains to be determined whether other newcomers to the superfamily of tubulins contain sites prone to posttranslational modification.     

Immunocytochemical study of tubulin in the 9+‘1’ sperm axoneme of a monogenean (Platyhelminthes), Pseudodactylogyrus sp.

The spermatozoon of the monopisthocotylean monogenean Pseudodactylogyrus sp. (a gill parasite of eels) has a single axoneme showing a 9+‘1’ pattern, a nucleus and a mitochondrion, but has no cortical microtubules. This species thus provides a very simple model for the study of tubulin in the 9+‘1’ axonemes of the Platyhelminthes, in contrast with digenean sperm which have a more complex spermatozoon with two such axonemes and cortical microtubules. Indirect immunofluorescence labelling of tubulin shows that the elongating spermatids, initially lying in all directions in the early stages, are arranged as parallel elements in further stages. The number of spermatids in an isogenic group could also be precisely counted and equals 32. Nuclear labelling with fluorescent dyes shows that the nuclei, first located in the common mass of the spermatids, later elongate and migrate into the growing spermatids, and that the nucleus is located in the central part of the mature spermatozoon, with the two extremities devoid of nucleus. Labelling with antibodies directed against acetylated, tyrosinated, and polyglutamylated tubulin gave positive results, thus indicating that these post-translational modifications of tubulin are present in the axoneme of spermatids and spermatozoa of monopisthocotylean monogeneans.     

Posttranslational tyrosination/detyrosination of tubulin

Tubulin can be posttranslationally modified at the carboxyl terminus of the alpha-subunit by the addition or release of a tyrosine residue. These reactions involve two enzymes, tubulin: tyrosine ligase and tubulin carboxypeptidase. The tyrosine incorporation reaction has been described mainly in nervous tissue but it has also been found in a great variety of tissues and different species. Molecular aspects of the reactions catalyzed by these enzymes are at present well known, especially the reaction carried out by the ligase. Several lines of evidence indicate that assembled tubulin is the preferred substrate of the carboxypeptidase, whereas nonassembled tubulin is preferred by the ligase. Apparently this posttranslational modification does not affect the capacity of tubulin to form microtubules but it generates microtubules with different degrees of tyrosination. Variation in the content of the carboxyterminal tyrosine of alpha-tubulin as well as changes in the activity of the ligase and the carboxypeptidase are manifested during development. Changes in the cellular microtubular network modify the turnover of the carboxyterminal tyrosine of alpha-tubulin. Different subsets of microtubules with different degrees of tyrosination have been detected in interphase cells and during the mitotic cycle. Data from biochemical, immunological, and genetic studies have been compiled in this review; these are presented, with pertinent comments, with the hope of facilitating the comprehension of this particular aspect of the microtubule field.     

F-Actin and Tubulin During Spermatogenesis in Gamasid Mites (Acari: Parasitina)

Abstract F-actin and tubulin behaviour was investigated using fluorescence probes and electron microscopy in the course of spermatogenesis in two gamasid mites, Porrhostaspis lunulata Müller (Parasitidae) and Pergamasus truatellus Athias-Henriot (Pergamasidae). In spermatogonia and primary spermatocytes of both species, the proteins were localized mainly in the intercellular bridges and, in lesser quantities, in the cytoplasm. Overall, actin was present along the plasma-lemmal contact sites of the gonial cells. At the beginning of spermatid elongation, actin could be detected in two regions: in perinuclear cytoplasm and under the plasmalemma. Subplasmalemmal actin, visible as threads running along acrosome-adhering protrusions of the nuclear envelope, is supposedly located within the electron-dense material filling the subacrosomal gap. Tubulin was found on both sides of each actin thread; its location was consistent with two sets of microtubules adhering to the inner acrosomal membrane. Their involvement in acrosome shaping is suggested. As spermatid elongation terminated, the previous pattern of proteins disappeared. In Pergamasus, however, actin emerged briefly near the centrifugal ends of spermatids (granular bodies zone). In spermatocyte-containing cysts, actin and tubulin fluorescence (more pronounced in Porrhostaspis) was associated with intercellular junctions between the cyst cells. In both species, diffuse actin fluorescence was also detected in the cytoplasm of cyst cells assembling elongated spermatids; the reaction was intensified at the end of the elongation process, when the cytoplasm of cyst cells aggregated around the centripetal ends of spermatids.     

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

Microtubular system during spermiogenesis and in the spermatozoon of Convoluta saliens (platyhelminthes,acoela): Tubulin immunocytochemistry and electron microscopy

Spermiogenesis and the spermatozoon were studied in Convoluta saliens, an acoel platyhelminth, by transmission electron microscopy, labelling of nuclei and immunocytochemistry of tubulin with various antibodies. Spermiogenesis involves formation of a long spermatid shaft containing two axonemes. It is established that the nucleus, after a stage of elongation, does not migrate up to the distal extremity of the spermatid, and that the centriolar derivatives are located at the distal extremity of the shaft. This contrasts with the parasitic Platyhelminthes. The mature spermatozoon, 180 μm in length, comprises a nuclear region, 50 μm in length, and a cytoplasmic region, with a short region of overlap. The cytoplasmic region contains two lateral axonemes with a 9 + 2 pattern of microtubules, granules of two different sizes, and two rows of longitudinal microtubules in the center. Each row consists of 5–6 singlet microtubules, with links between them. Whereas the two axonemes are labelled by antibodies against alpha, acetylated‐alpha, and beta tubulin, the microtubule rows are labelled only by the anti‐beta‐tubulin antibody. This suggests that acetylation does not occur in this part of the cytoskeleton, and that the epitope recognized by the anti‐alpha‐tubulin antibody (DM1A) is different in these units. Mol. Reprod. Dev. 52:74–85, 1999. © 1999 Wiley‐Liss, Inc.     

The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Immunoelectron microscopical detection of tubulins during spermiogenesis in phytophagous bugs (Hemiptera: Pentatomidae)

Summary

Ultrastructural and immunocytochemical studies were carried out in the tail region of spermatids and spermatozoa of the phytophagous bugs, Acrosternum aseadum and Euchistus heros. The axoneme presented a 9+9+2 microtubule pattern and bridges occurred between axonemal microtubules 1, 5, and mitochondrial derivatives. Two paracrystalline structures, embedded in an amorphous matrix, were observed in the mitochondrial derivatives. The axonemal microtubules contained alpha, acetylated and tyrosinated tubulin. Cytoplasmic microtubules contained alpha, beta and gamma tubulin. Moreover, the gamma tubulin was detected near the electron dense rod, an element associated with the centriole, suggesting that this structure may be a microtubule organizing center.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Ultrastructure of distal flagellar swellings in spermatocytes and spermatids of Ephestia kuehniella Z. (Pyralidae,Lepidoptera)

Summary Development of flagella was investigated by transmission electron microscopy in spermatocytes and spermatids of the Mediterranean mealmoth, Ephestia kuehniella Z. Growing flagella displayed voluminous distal swellings. In short flagella the apical portion of the swellings contained an amorphous, dense accumulation. In more developed flagella a less dense proximal extension of the apical accumulation was formed, which in turn was in contact with the elongating flagellar microtubules. The material of the flagellar tip is interpreted as being a precursor of the axoneme containing mainly tubulin. The material may be converted into the axoneme.     

Centriole Disassembly In Vivo and Its Effect on Centrosome Structure and Function in Vertebrate Cells

Glutamylation is the major posttranslational modification of neuronal and axonemal tubulin and is restricted predominantly to centrioles in nonneuronal cells (Bobinnec, Y., M. Moudjou, J.P. Fouquet, E. Desbruyères, B. Eddé, and M. Bornens. 1998. Cell Motil. Cytoskel. 39:223–232). To investigate a possible relationship between the exceptional stability of centriole microtubules and the compartmentalization of glutamylated isoforms, we loaded HeLa cells with the monoclonal antibody GT335, which specifically reacts with polyglutamylated tubulin. The total disappearance of the centriole pair was observed after 12 h, as judged both by immunofluorescence labeling with specific antibodies and electron microscopic observation of cells after complete thick serial sectioning. Strikingly, we also observed a scattering of the pericentriolar material (PCM) within the cytoplasm and a parallel disappearance of the centrosome as a defined organelle. However, centriole disappearance was transient, as centrioles and discrete centrosomes ultimately reappeared in the cell population.During the acentriolar period, a large proportion of monopolar half-spindles or of bipolar spindles with abnormal distribution of PCM and NuMA were observed. However, as judged by a quasinormal increase in cell number, these cells likely were not blocked in mitosis.Our results suggest that a posttranslational modification of tubulin is critical for long-term stability of centriolar microtubules. They further demonstrate that in animal cells, centrioles are instrumental in organizing centrosomal components into a structurally stable organelle.     

Distribution and Characteristics of βII Tubulin-Enriched Microtubules in Interphase Cells

We have used a polyclonal antibody (Ab196) that specifically recognizes the βII tubulin isotype to examine the subcellular distribution and properties of microtubules enriched in this isotype. Antibody specificity was tested by a method that involves the analysis of its interaction with individual β isotypes. Using photoimaging analysis, we observed βII tubulin-enriched microtubules in the perinuclear region, as well as in the microtubules close to the periphery of interphase cells. The observed sorting of βII-enriched microtubules together with the reported increased levels of βII tubulin in taxol-resistant cells (M. Haberet al.,1995,J. Biol. Chem.270, 31269–31275) prompted us to study the behavior of microtubules enriched in this isotype after different depolymerizing treatments. After cold or nocodazol treatments, βII-enriched microtubules anchored at the centrosome and at the cell periphery were observed. In addition, cold-resistant microtubules were marked mainly by the specific anti-βII tubulin antibody but not by anti-acetylated α tubulin, suggesting the presence of different stable microtubule subsets enriched in particular tubulin isoforms.     

Posttranslational modification and microtubule stability

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.     

Interaction of captan with mammalian microtubules

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.     

Posttranslational modifications of alpha tubulin: detyrosination and acetylation differentiate populations of interphase microtubules in cultured cells

Subsets of microtubules enriched in posttranslationally detyrosinated (Gundersen, G. G., M. H. Kalnoski, and J. C. Bulinski. 1984. Cell. 38:779) or acetylated (Piperno, G., M. Le Dizet, and X. Chang. 1987. J. Cell Biol. 104:298), alpha tubulin have previously been described in interphase cultured cells. In this study an immunofluorescence comparison of these minor populations of microtubules revealed that, in African green monkey kidney epithelial cells (TC-7 line), the population of microtubules enriched in detyrosinated tubulin was virtually coincident with the population enriched in acetylated alpha tubulin. In some cell types, however, such as human HeLa or marsupial PtK-2 cells, only one posttranslationally modified form of tubulin, i.e., acetylated or detyrosinated, respectively, was detectable in microtubules. In TC-7 cells, although both modifications were present, dissimilar patterns and kinetics of reappearance of microtubules enriched in detyrosinated and acetylated tubulin were observed after recovery of cells from microtubule-depolymerizing treatments or from mitosis. Thus, a minor population of microtubules exists in cultured cells that contains an elevated level of tubulin modified in either one or two ways. While these two modifications occur primarily on the same subset of microtubules, they differ in their patterns of formation in vivo.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

Structural organization and distribution of the symbiotic bacteria <Emphasis Type="Italic">Wolbachia</Emphasis> during spermatogenesis of <Emphasis Type="Italic">Drosophila simulans</Emphasis>

Electron microscopy and morphometric analysis have shown that the symbiotic bacteria Wolbachia occur the testis cells D. simulans during spermatogenesis and are absent in mature spermatids. Bacteria did not affect the structural organization of testis cells, which have a typical morphology during morphogenesis. Bacteria were distributed along the meiotic spindle microtubules near the mitochondria. They increased in number in spermatids at the stage of elongation. Endosymbionts aggregated at the spermatid distal end and contained many vacuoles but were absent at the spermatid proximal end near the nuclei. It was shown for the first time that the diameter of spermatids in a strongly infected line was two of three times that in a noninfected line. We hypothesize that the increase in the number of endosymbionts during spermatid elongation can affect the chromatin condensation in the spermatozoon.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 41–50.Original Russian Text Copyright © 2005 by Dudkina, Kiseleva.     

Post-translational tubulin modifications in spermatogeneous cells of the pteridophyteCeratopteris richardii

Summary Acetylation and tyrosinization are post-translational modifications of tubulin generally associated, respectively, with highly stable or dynamic microtubule arrays in animals and protists. Little is known of these modifications in land plants, however. We examined the presence and distribution of post-translational tubulin modifications in developing spermatogenous cells of the pteridophyteCeratopteris richardii by immunofluorescence and immunogold, utilizing antibodies specific for acetylated and tyrosinated tubulin. Acetylated tubulin is found in mid to late stage spermatogenous cells in stable microtubule configurations: the spline, flagella, and basal bodies. Tyrosinated tubulin, a modification associated with dynamic microtubule arrays, is also present in these structures as well as all other microtubules in the cell. The lamellar strip of the multilayered structure, a body previously described as tubulin-containing, was not labelled by any of the tubulin antibodies or antiserum. Treatment of cultures with the microtubule stabilizer taxol results in the appearance of new arrays of microtubules, including bundles in the cytoplasm. Only those new taxol-induced microtubule arrays present in mid to late stage cells (i.e., those with other normally acetylated tubulin arrays) have acetylated domains. Younger spermatogenous cells had similar microtubule bundles but no acetylated tubulin. Tyrosinated tubulin was found in all these taxol-stabilized arrays. These data indicate that, although these pteridophyte cells have the ability to acetylate tubulin, that this ability is limited to stages after the final spermatogenous cell mitosis and is limited to the highly stable spline and flagella microtubules.Abbreviations LS lamellar strip of multilayered structure - MTOC microtubule organizing center     

Molecular determinants for α-tubulin methylation by SETD2

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.