Journal: Nanoscale Advances

Article Title: Simultaneous activation and release of a microtubule inhibitor from amphiphilic polymeric nanoparticles

doi: 10.1039/d6na00763e

Figure Lengend Snippet: Representative lowest energy docking poses of (a) cis -PST-1, (b) trans -PST-1, (c) cis -proPST-1, and (d) trans -proPST-1 within the colchicine binding pocket of tubulin (PDB ID: 5LYJ). Ligands are represented as orange sticks, while interacting amino acid residues are shown as yellow sticks and labelled using single-letter amino acid notation. Hydrogen-bonding and hydrophobic interactions are indicated by red and blue dashed lines, respectively, with corresponding interaction distances (Å) shown. Structures and interaction maps were generated using PyMOL v3.0.

Article Snippet: Tubulin MAP-rich (porcine brain) (Cat. #ML116), Tubulin protein (fluorescent HiLyte 488): porcine brain (Cat. #TL488M), General tubulin buffer (Cat. #BST01), Tubulin glycerol buffer (Cat. #BST05) and Taxol (Cat. #TXD01) and GTP (Cat. #BTS06) were obtained from Cytoskeleton, Inc and used as per the instruction manual.

Techniques: Docking Assay, Binding Assay, Generated

Journal: Nanoscale Advances

Article Title: Simultaneous activation and release of a microtubule inhibitor from amphiphilic polymeric nanoparticles

doi: 10.1039/d6na00763e

Figure Lengend Snippet: (a) Schematic representation of light-controlled inhibition of microtubule polymerisation by proPST-1-loaded polymer nanoparticles (proPST-1@PN). Under dark conditions, the encapsulated photocaged prodrug remains inactive, allowing normal microtubule formation. Upon 365 nm irradiation, photolytic uncaging and release of active cis -PST-1 suppress microtubule assembly. Created in BioRender. Singh, H. (2026) https://BioRender.com/fac5r0eT . (b) Tubulin polymerisation kinetics monitored by absorbance at 340 nm for control samples, empty polymer nanoparticles (PN control), and proPST-1@PN under dark and irradiated conditions. Irradiation of proPST-1@PN results in pronounced inhibition of tubulin polymerisation, whereas no inhibition is observed in the dark. (c) Fluorescence microscopy images of microtubule structures formed in the presence of control samples, proPST-1@PN under dark conditions, and irradiated proPST-1@PN. Normal microtubule growth and network formation are observed in control and dark conditions, while irradiation results in substantial disruption of microtubule assembly. Scale bars: 10 µm.

Article Snippet: Tubulin MAP-rich (porcine brain) (Cat. #ML116), Tubulin protein (fluorescent HiLyte 488): porcine brain (Cat. #TL488M), General tubulin buffer (Cat. #BST01), Tubulin glycerol buffer (Cat. #BST05) and Taxol (Cat. #TXD01) and GTP (Cat. #BTS06) were obtained from Cytoskeleton, Inc and used as per the instruction manual.

Techniques: Inhibition, Polymer, Irradiation, Control, Fluorescence, Microscopy, Disruption

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Ana1 is essential for centriole duplication and promotes centriole overelongation in fly spermatocytes. (A) Schematic representation of the two independent ana1 null alleles, denoted as ana1 Δa and ana1 Δb , created by CRISPR/Cas9 mutagenesis. Both mutants had deletion of the entire ana1 coding region. These alleles were then crossed to generate the ana1 null mutant background (referred to as ana1 −/− ) used throughout this study. (B) Western blot of extracts from 50 to 60 testes of indicated genotypes. Detection of Ana1 was performed using specific antibodies, with α-tubulin as a loading control for protein levels in lysates. (C) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), Cep135-mRuby3 (red), and DNA (blue). Scale bars, 10 µm (overview image) or 2 µm (insets). (D) Graphs show the mean ± SD quantification for centriole lengths in mature primary spermatocytes, using Asl as a centriole marker for each genotype. Each point on the graph represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. **** P < 0.0001. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: CRISPR, Mutagenesis, Western Blot, Control, Staining, Marker

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Mapping of the interaction region between Cep135 and Ana1. (A) Climbing ability was assessed by measuring the percentage of flies per genotype that climbed past the 5-cm mark within 2 s. Each data point represents the percentage from 20 flies, with three independent biological replicates using distinct cohorts of flies per genotype (total of 60 flies per genotype). Error bars indicate the mean ± SD. **** P < 0.0001 (unpaired two-tailed Student’s t test). (B) Centriole pairs, marked by Asl localization, were quantified in 15–20 cells per testis from the indicated genotypes. Each data point represents the number of centriole pairs per testis, derived from five independent testes per genotype. Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (C) Pull-down of EGFP-Ana1 from S2 cells incubated with testis lysates (150 testes/genotype) from w 1118 or cep135 c04199 mutants. Blots were probed with anti-Cep135 (aa1–225). α-Tubulin is a loading control. The residual Cep135 in mutants (Cep135-N*) fails to bind Ana1. (D and E) ITC-based experiments to screen for interactions between Cep135 and Ana1 fragments, and a schematic representation showing various Ana1 and Cep135 constructs analyzed for binding in this study. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: Two Tailed Test, Derivative Assay, Incubation, Control, Construct, Binding Assay

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Cep135 is required for Ana1-induced centriole overelongation. (A) Western blot of extracts from 50 to 60 testes of indicated genotypes. Detection of Cep135 was performed using specific antibodies, with α-tubulin as a loading control for protein levels in lysates. (B) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), and DNA (blue). Scale bars, 10 µm (overview image) or 2 µm (insets). (C) Graphs show the mean ± SD quantification for centriole lengths in mature primary spermatocytes, using Asl as a centriole marker for each genotype. Each point on the graph represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. **** P < 0.0001, ** P < 0.01. (D) Graphs show the mean ± SD quantification of Ana1 mean fluorescence intensity (per unit length of centriole) for each genotype. Each point on the graph represents the average intensity measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ns, not significant using a two-tailed unpaired t test. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: Western Blot, Control, Staining, Marker, Fluorescence, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Ana1-Cep135 interaction is essential for centriole length control in primary spermatocytes. (A) Western blot of extracts from 50 to 60 testes of indicated genotypes. Immunoblot analysis of Ana1 protein variants with an anti-Ana1 primary antibody. α-Tubulin is shown as a loading control. (B) Graphs showing the male fertility of different genotypes, as measured by the number of progeny pupae produced from mating with WT ( w 1118 ) virgin females. ** P < 0.01, *** P < 0.001. (C and D) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), Cep135-mRuby3 (red), and DNA (blue). Scale bar, 10 µm (overview image) or 2 µm (insets). (E) Graphs showing the mean ± SD quantification for centriole lengths in mature primary spermatocytes, using Ana1, Asl, or Cep135 as a centriole marker for each genotype. Each point on the graph represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ** P < 0.01, *** P < 0.001, **** P < 0.0001. (F) Graphs showing the mean ± SD quantification of Ana1 or Cep135 mean fluorescence intensity (per unit length of centriole) for each genotype. Each point on the graph represents the average mean intensity measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. * P < 0.05. ns, not significant. (G) TEM of EGFP-Ana1 (top panels) and EGFP-Ana1 Δ244–371 (bottom panels) in cross-sections (left) and longitudinal sections (right) of centrioles. Cross-sectional views reveal that the ninefold symmetric centriole architecture is preserved in both WT and mutant flies analyzed. Longitudinal sections show that full-length EGFP-Ana1 labels normal-length centrioles (top right), whereas deletion of the residues 244–371 results in shortened centrioles (bottom right). For each genotype, centrioles were examined from at least three independent testes, with a total of 8–12 centrioles analyzed. Scale bar, 200 nm. WT, wild type. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: Control, Western Blot, Produced, Staining, Marker, Fluorescence, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Ana1 binds to and bundles MTs in vitro . (A and B) Coomassie blue–stained SDS-PAGE gel of a MT cosedimentation experiment performed with 2 mg/ml polymerized tubulins and different Ana1 fragments. The experiment was repeated three times with similar results. (C and D) Cartoon representations for AF3-predicted Ana1 180–497 or Ana1 1404–1729 structural models showing the distribution of surface-exposed positive charges. Residues subjected to mutation are highlighted in the zoomed-in view boxes. (E) Confocal images of in vitro Taxol-stabilized MTs at 1 μM (ratio of rhodamine-labeled tubulin/unlabeled tubulin is ∼10%; red) mixed with 1 μM Ana1 180–497 or 2 μM Ana1 1404–1729 protein. Scale bar, 20 µm. Identical imaging settings were used for all groups. Note that an inset is included for tubulin-only control, showing images of rhodamine-labeled tubulin with different contrasts to the protein–tubulin mixture group for better visualization. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: In Vitro, Staining, SDS Page, Mutagenesis, Labeling, Imaging, Control

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: Ana1-MT interaction is essential for Ana1-mediated centriole elongation in primary spermatocytes. (A) Western blot of extracts from 50 to 60 testes for indicated genotypes. Immunoblot analysis of Ana1 protein variants with an anti-Ana1 primary antibody. α-Tubulin is shown as a loading control. (B) Graphs showing the male fertility of different genotypes, as measured by the number of progeny pupae produced from mating with WT ( w 1118 ) virgin females. **** P < 0.0001. ns, not significant. (C and D) Centrioles in primary spermatocytes stained to reveal EGFP-Ana1 (green), Asl (red), Cep135-mRuby3 (red), and DNA (blue). Scale bar, 10 µm (overview image) or 2 µm (inset image). (E) Graphs showing the mean ± SD quantification for Ana1 and Cep135 mean intensity per unit length of centrioles in primary spermatocytes. Each point on the graph represents the average mean intensity measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. ns, not significant. (F) Graphs showing the mean ± SD quantification for centriole lengths in mature primary spermatocytes using Ana1, Asl, or Cep135 as a centriole marker for each genotype. Each point on the graphs represents the average length measured from a 16-cell cyst with 10–15 centrioles scored; five independent testes were measured for each genotype with 2–3 cell cysts scored in each testis. * P < 0.05, ** P < 0.01, **** P < 0.0001. ns, not significant. (G) Cross-sections (left) of centrioles in Drosophila primary spermatocytes by TEM showing preserved ninefold symmetry in both EGFP-Ana1 (top) and EGFP-Ana1-RKNC (bottom) groups. Longitudinal sections (right) show that the RKNC mutation leads to centriole shortening compared with full-length Ana1 controls. For each genotype, centrioles were examined from at least three independent testes, with a total of 8–12 centrioles analyzed. Scale bar, 200 nm. WT, wild type. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: Western Blot, Control, Produced, Staining, Marker, Mutagenesis

Journal: The Journal of Cell Biology

Article Title: Ana1/Cep295 regulates centriole elongation via Cep135 and microtubules

doi: 10.1083/jcb.202504094

Figure Lengend Snippet: MT binding–deficient Ana1 mutants fail to induce centriole overelongation. (A) Representative images showing centriole elongation in primary spermatocytes at various stages from different genotypes, stained for EGFP-Ana1 (green) and Asl (red). Scale bar, 2 µm. (B) Centriole pairs, marked by Asl localization, were quantified in 15–20 cells per testis from the indicated genotypes. Each data point represents the number of centriole pairs per testis, derived from five independent testes per genotype. Error bars indicate the mean ± SD. ns, not significant (unpaired two-tailed Student’s t test). (C) In vitro GST pull-down assay showing interactions between MBP-Ana1 1404–1729 or Ana1 1404–1729RK and GST-PBD when incubated with or without PLK (indicated as “Plk1 +/−”). Top panel, western blot analysis of MBP-Ana1 being pulled down by GST-PBD. Note that the binding was enhanced by PLK1 phosphorylation and the RK mutation had no effect on protein binding. Middle panel, SDS-PAGE analysis showing comparable amount of GST-PBD coupled to the GST beads. Bottom panel, SDS-PAGE analysis showing MBP-Ana1 input. Asterisks highlight the degradation band of MBP-Ana1. (D) Coomassie blue–stained SDS-PAGE gel of a MT cosedimentation experiment performed with 2 mg/ml polymerized tubulins and His-Ana1 1–497 or His-Ana1 139–497 (CR1 deletion). (E) Rhodamine-labeled tubulin (red; ∼10% labeling ratio with unlabeled tubulin) was mixed with 1 μM Taxol-stabilized MTs in combination with 1 μM Cep135 1–498 , 1 μM Ana1 180–497 , 1 μM Ana1 Δ282–301 , or 1 μM Ana1 RKN as indicated. Identical imaging settings were used across all groups for direct comparison. Scale bar, 20 μm. PBD, Polo-box domain. Source data are available for this figure: .

Article Snippet: For confocal imaging, 1 μl of rhodamine-labeled tubulin (Cat. No. TL590M; Cytoskeleton) was added to the tubulin preparation.

Techniques: Binding Assay, Staining, Derivative Assay, Two Tailed Test, In Vitro, Pull Down Assay, Incubation, Western Blot, Phospho-proteomics, Mutagenesis, Protein Binding, SDS Page, Labeling, Imaging, Comparison