porcine tubulin protein  (Cytoskeleton Inc)

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: Low‐magnification images for the analyses of the interaction between PSDLs and MTs. (A, B) Time course of MT polymerization in the presence or absence of PSDLs. Tubulin polymerization (ML116, 2 mg/mL) was performed in the presence (A) or absence (B) of PSDLs. The PSDL‐laden membrane was blocked at 0°C and 37°C before contacting with tubulin‐containing droplets (see Figure for the effect of blocking the Formvar membrane). PSDL‐laden grids were contacted with tubulin‐containing droplets at 0°C and then transferred to a pre‐heated aluminum block at 37°C to initiate tubulin polymerization. The PSDL‐unladen grid membrane was not blocked and brought into contact with tubulin‐containing droplets for 30 s at specific time points. “Before” samples in the “Tubulin+PSDL” experiment were not put in contact with tubulin‐containing droplets. Samples on the grids were fixed for 5 min and negatively stained with nano‐W. (C, D) Quantification of changes in particle size. Experiments in the presence (C) or absence (D) of PSDLs were performed as stated above. EM images of the negatively stained specimens were captured randomly at an 8000× magnification. Particles of different sizes (> 100 nm) were counted manually, as it was not possible to simultaneously measure structures of varying sizes within the same visual field under identical contrast conditions using ImageJ for particle analysis. The vertical line represents the number of particles in a field imaged at 8000× magnification. The number of 8000× magnified images (n) and the total number of particles (p.n.) are indicated at the bottom. Significance was tested using Student's t test or the Mann–Whitney's U test. Of note, the numerical comparison of (C) and (D) is invalid because the conditions for protein trapping in the Formvar membrane differed between the two systems. (E) Negative‐staining EM images of PSDLs before contact with tubulin‐containing droplets. (F) Negative‐staining EM images of PSDLs after incubation with MTs subjected to the polymerization–depolymerization cycle. Clearly visible meshwork structures are indicated by arrows.

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Membrane, Blocking Assay, Staining, Particle Size Analysis, MANN-WHITNEY, Comparison, Negative Staining, Incubation

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: PSDL‐like structures contained in purified tubulin preparations. Examples of standard structures of PSDLs (A‐a and A‐b) and PSD (OG12) (A‐c) examined by negative‐staining EM. (B, C) Structures in MAP‐rich tubulin (ML116) before (B) and during polymerization at 37°C for 5 min (C), examined by negative‐staining EM. (D) Negatively stained pure tubulin (> 99% purity, T240) before (D‐a) and during polymerization at 37°C for 15 min (D‐b,c). The size distribution of the particles in T240 tubulin is shown in (D‐d). The vertical line represents the number of particles in a field imaged at 8000× magnification. The number of 8000× magnified images (n) and the total number of particles (p.n.) are indicated in the graph. Particles with a maximum diameter exceeding 100 nm were counted. (E, F) Negatively stained structures contained in the T240 tubulin before (E) and during polymerization at 37°C for 15 min (F). Both tubulin preparations labeled as “before polymerization” were suspended in the same buffer used for polymerization. All samples were fixed with 1% (v/v) glutaraldehyde prior to negative staining. An arrow labeled “sm” in (F‐e) indicates a small structure at the MT end (see also Figure for enlarged images that better illustrate their morphological differences).

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Purification, Negative Staining, Staining, Labeling

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: Investigation of the interaction between tubulin/MTs and PSDL/PSD using non‐EM‐based methods. (A) Co‐sedimentation analysis. Binding of tubulin to the PSD (OG12). Pure tubulin (T240, 5 μg) was mixed with OG12 (approx. 1 μg) and incubated for 20 min at 37°C. The samples were centrifuged, washed once, and the final pellets were analyzed by SDS‐PAGE (A‐a). Tubulin alone and OG12 alone were processed in the same way for comparison. The areas of the tubulin bands in lanes 2, 3, and 4 (gray) shown in (A‐b) were quantified, and the results are presented in the graph (A‐c). (B–D) Experiments using latex beads. (B) Entrapment of polymerizing MTs into immobilized latex beads. Latex bead‐immobilized EM grid was contacted to the solution in which MT (ML116) was polymerized. The grids were fixed, negative‐stained after 5 min of MT polymerization, and observed by EM. (B‐a, B‐b) Typical examples of the latex beads (LB) on the EM grids without or associated with MTs. Arrowheads indicate MT. (B‐c) The same experiment was carried out using a PSDL‐immobilized EM grid. Graph showing the amount of grid‐immobilized beads or PSDLs associated with MTs. The bars show mean values (mean ± SE) of three quantifications, which included 85, 108, and 75 beads, and 310, 355, and 305 PSDLs. (C, D) Tubulin (ML116) and latex beads (C) or PSD (D) were mixed in a solution, and MT polymerization was induced at 37°C for 5 min. They were captured on an unblocked EM grid, negatively stained, and observed using electron microscopy (EM) at 600× magnification. Beads alone were incubated and treated in the same manner as a control. The number of beads trapped on the grid was counted manually, and the quantification results are presented as the number of beads in a single square of a 400‐mesh EM grid (C‐c). Most dark spots in (C‐a) contained a single bead, while some contained clustered beads, as shown in the magnified area marked with an asterisk (C‐a). Only three beads are trapped in the field shown in (C‐b), as indicated by the arrows. The spot marked with an open arrowhead does not contain a bead. The dark background in (C‐b) is due to polymerized MT networks trapped on the grid. Tubulin (ML116) and PSD (OG12) were mixed in a solution, followed by the procedure described in (C), except for the EM imaging, which was performed at 8000× magnification. The field area in the graph corresponds to images obtained at this magnification. The number of entrapped beads was significantly reduced upon incubation with polymerizing tubulin, as shown in (C‐c), while the number of entrapped PSDs remained unaffected (D‐c). Arrows in (D) indicate PSDs, as confirmed by the enlarged views shown in (D‐a, 1, and 2). Statistical significance was assessed using a two‐tailed Student's t test, with the p ‐value indicated in parentheses. fn, field number counted.

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Sedimentation, Binding Assay, Incubation, SDS Page, Comparison, Staining, Electron Microscopy, Control, Imaging, Two Tailed Test

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: Distribution of PSDL‐like structures on MTs. Tubulin (ML116) was polymerized in the presence of PSDLs for 5–6 min as described in the legends of Figures and . In this article, structures that resemble PSDLs in morphology and size, including the original PSDLs, are collectively referred to as “PSD‐like structures.” This is because distinguishing between them is extremely difficult. (A–F) Various types of associations between polymerizing MTs and PSDL‐like structures. To enhance clarity, MTs are indicated using closed arrowheads in low‐contrast photos. MTs are not labeled in (F‐a) because there are several MTs distributed in a complicated manner. Free MT ends are indicated with open arrowheads marked with F. Clearly identifiable contact sites between PSDL‐like structures and MTs are indicated with arrows. The image in (F‐e) is a magnified view of the rectangular region shown in (F‐d). PSDL‐like structures marked with asterisks connect MTs, particularly the one in (F‐e), which links MTs labeled I and II (F‐d and F‐e). The distribution frequency of PSDL‐like structures is shown in (G). The distribution of PSDL‐like structures on MTs was analyzed by capturing images of full‐length MTs at 6000–10 000× magnification and viewing the magnified images on a computer display. The frequency is presented as a percentage of the total PSDL‐like structures counted (118 PSDL‐like structures > 200 nm, as described in the Methods section). MTs exhibiting multisite‐type binding were excluded from the analysis due to the complex crowding of MTs. Structures distributed in the non‐end MT region, typically those smaller than 200 nm as indicated by the open arrow in (D), are also present in the tubulin preparation; therefore, they were excluded from this graph.

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Labeling, Binding Assay

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: EM observation of contact points between polymerizing MTs and PSDL‐like structures. Tubulin (ML116) was polymerized in the presence of PSDL for 5 min, as described in the figure legends for Figures and . Images focused on structures associated with MTs and their contact points between polymerizing MTs. The different types of associated structures and interactions are tentatively grouped and displayed in panels (A–F). Panel (G) shows the quantification of MT ends modified by associations with characteristic structures, as illustrated in panel (E). Samples were prepared, examined, and displayed as described in the legend for Figure . The percentage of PSDL‐associated MT ends represents the average of three independent experiments, which included 179, 55, and 155 MT ends in the “tubulin alone” samples, and 162, 119, and 169 MT ends in the “tubulin + PSDL” samples. Statistical significance was determined using a two‐tailed Student's t test. Direct connections (d.c.) between PSDL‐like structures and the MT end are indicated by small open arrows. Multiple arrows indicate tapered or enlarged MT end regions. Free MT ends are denoted by open arrowheads marked with “F”. MTs that are difficult to visualize due to low contrast are highlighted with multiple closed arrowheads. Characteristic mesh‐like structures associated with the MT non‐end regions are indicated by large open arrows (F). The images in panels (D‐a) and (D‐b) are magnified views of the lower half of Figure and the white square in panel (Figure ‐a), respectively.

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Modification, Two Tailed Test

Journal: Journal of Neurochemistry

Article Title: Tubulin and GTP Are Crucial Elements for Postsynaptic Density Construction and Aggregation

doi: 10.1111/jnc.70085

Figure Lengend Snippet: Identification of tubulin through immuno‐gold negative staining EM in GTP‐treated TX‐PSD. TX‐PSD treated with 10 mM GTP at 0°C for 90 min was transferred to a Formvar membrane on the EM grid, fixed with 0.25% glutaraldehyde at 0°C for 5 min, labeled with anti‐tubulin polyclonal antibodies, followed by gold (10 nm)‐labeled secondary antibodies, and negatively stained. Fixation immediately after treatment with GTP was necessary to prevent the loss of short fibers produced following treatment with GTP. Blockade of the specimens was omitted as this was found to be unnecessary. (A‐a) Tubulin immunoreactivity (IR) on the short fibers produced following the treatment of TX‐PSD with GTP. Tubulin‐immunoreactive gold particles are indicated with white arrows. (A‐b), (A‐c) Negative control (NC) prepared by treating TX‐PSD with 10 mM GTP or MTs derived from ML116 tubulin, polymerized through incubation at 37°C for 5 min. Specimens were treated as described in (A‐a), but without the use of primary antibodies (see Figure for positive and negative control, respectively, consisting of MTs generated from ML116). Enlarged images of the boxed areas in (A‐b) and (A‐c) are shown in (A‐d), (A‐e), and (A‐f). The fine structures of short fibers produced through treatment with GTP typically consisted of characteristic bundled bead‐like structures, which were different from those of normal MT fibers. Black arrows in (A‐d) and (A‐e) indicate incomplete bead‐like structures in normal MT fibers, suggesting commonality between the two fibers, that is, short fibers were derived from MTs or formed in a similar way as MTs. (B) Tubulin IR on the fiber structures remaining in TX‐PSD aggregates following treatment with 10 mM GTP. Tubulin‐immunoreactive gold particles are indicated by black arrows in (B‐a), (B‐b), (B‐c), and (B‐d), except for those in the inserts. Immuno‐gold particles in (B‐e, B‐f, and B‐g) are not indicated because their distribution was locally abundant. The enlarged views of the boxed portions are shown in the inserts to depict the bead‐like structures seen in (A‐b). (B‐h) Negative control for the immuno‐gold detection experiment, in which primary antibodies were omitted, showing the absence of immuno‐gold particles (see also Figure for immuno‐gold labeled tubulin in the TX‐PSDs during and after interaction with polymerizing MTs).The detailed structures of the short fibers, as well as those of the remaining fibers associated with PSD aggregates, are shown in Figure . The fibers were bundled into three or four strands consisting of connected ring structures with a typical diameter of 12.6 nm each (Figure ). The detailed structures of negatively stained normal MTs (Figure ) were different from those of short fibers. Similar but imperfect ring‐like structures were occasionally observed in normal MTs (arrows in Figure ), and were not regularly aligned in a line. The presence of imperfect ring‐like structures in normal MTs suggested a similarity between the short fibers and normal MTs. Thus, the short fibers may have been derived from MTs or formed in a similar manner as MTs. However, the molecular organization of the short fibers appeared to be slightly different or changed from that of normal MTs.

Article Snippet: Tubulin MAP‐rich , ML116 , Cytoskeleton Inc. (Denver, CO, USA).

Techniques: Negative Staining, Membrane, Labeling, Staining, Produced, Negative Control, Derivative Assay, Incubation, Generated