HMMR promotes neuronal morphogenesis.

(A) Representative images of hippocampal neurons co-transfected with the EGFP-expressing and the indicated shRNA-expressing plasmids on 0 DIV and fixed on 4 DIV. Neurons were immunofluorescence stained with the dendrite marker MAP2 and the axon marker SMI312 (top). Quantification of (B) total neurite length per neuron, (C) normalized total axon length per neuron, (D) normalized total dendrite length per neuron, and (E) normalized branch density (i.e., branch number per 50 µm of neurite). **** P<0.0001, one-way ANOVA followed by Dunnett’s post-hoc test. More than 20 neurons were analyzed per condition per repeat. (F) Representative images of hippocampal neurons transfected with AcGFP- or AcGFP-mHMMR-expressing plasmid on 0 DIV and fixed on 3 DIV. Neurons were immunofluorescence stained with the dendrite marker MAP2 and the axon marker SMI312. Quantification of (G) total neurite length per neuron, (H) normalized total axon length per neuron, and (I) normalized total dendrite length per neuron. **** P<0.0001, two- tailed Student’s t-test. More than 50 neurons were analyzed per condition per repeat. (J) Sholl analysis of the neurite branching complexity. ** P<0.01, two-way ANOVA followed by Sidak’s post-hoc tests. The solid line and shaded area indicate mean and s.e.m. collected from three independent repetitions (more than 50 neurons were analyzed per condition per repetition). All scale bars present 50 µm and all bar graphs are expressed as mean ± s.e.m. from three independent repetitions.

HMMR localizes to the microtubules in neurons.

(A) Representative images of 3 DIV hippocampal neurons directly fixed with formaldehyde (left) or permeabilized for 15 seconds before formaldehyde fixation (right). Neurons were immunofluorescence stained with antibodies against HMMR (top, pseudo-colored) and β-III-tubulin (middle, pseudo-colored). The merged image shows β-III-tubulin staining in red and HMMR in green. All images have the same scale and the scale bars present 50 μm. (B) Representative images of 4 DIV (top) and 7DIV (bottom) hippocampal neurons expressing AcGFP-mHMMR. Neurons were immunofluorescence stained with the β-III-tubulin antibody. AcGFP-mHMMR and β-III-tubulin signals were inverted to improve visualization. Red and white boxes at the soma are magnified in the insets. All images have the same scale and the scale bars present 50 μm. (C) Representative images of proximity ligation assay (PLA) on HMMR and β-III-tubulin in 3 DIV hippocampal neurons. The PLA image was inverted to improve visualization (left). DAPI was used to visualize the nuclei and brightfield microscopy was used to visualize the general appearance of neurons in the merged image (right). The scale bar presents 50 μm. (D) PLA puncta were present along the neurite shaft only when antibodies against HMMR and β-III-tubulin were both present. All images have the same scale and the scale bars represent 10 μm.

HMMR regulates microtubule stability in neurons.

(A) Representative images of 4 DIV hippocampal neurons expressing AcGFP-mHMMR. Neurons were immunofluorescence stained with the antibody against β-III-tubulin. White boxes at the soma and the neurite tip are magnified. Arrowheads indicate looped microtubules. (B) Representative pseudo-colored acetylated-α- tubulin-to-β-III-tubulin ratio images of non-targeting shRNA (top left panel) or Hmmr-targeting shRNA (top right panel) expressing 4 DIV hippocampal neurons. The transfection indicator EGFP signal was inverted to improve visualization (bottom panels). Only neurons possessing both β-III-tubulin and EGFP signals were quantified. Quantification of the acetylated-α-tubulin-to-β-III-tubulin intensity ratio in axon (C) and dendrite (D). ****, P<0.0001, two-tailed Mann-Whitney test. (E) Representative pseudo-colored acetylated-α-tubulin-to-β-III-tubulin ratio images of AcGFP-expressing control (top left panel) or AcGFP-mHMMR (top right panel) expressing 3 DIV hippocampal neurons. The AcGFP signal was inverted to improve visualization (bottom panels). Only neurons possessing both β-III-tubulin and AcGFP signals were quantified. Quantification of the acetylated-α-tubulin-to-β-III-tubulin intensity ratio within axon (F) and dendrite (G). ****, P<0.0001, two-tailed Mann-Whitney test. (H) Quantification of total neurite length per neuron in AcGFP- or AcGFP-mHMMR expressing 3 DIV hippocampal neurons treated with or without 100 nM nocodazole for 2 days. ****, P<0.0001, one-way ANOVA followed by Tukey’s post-hoc tests. All box plots are expressed as first quartile, median, and third quartile with whiskers extending to 5-95 percentile. More than 90 neurons were analyzed per condition per repeat. Scale bars represent 20 µm in (A) and 50 µm in (B) and (E).

HMMR regulates the dynamics of neuronal microtubules.

(A) Representative image of a 4DIV EB3-mCherry-expressing cortical neuron. The color boxes indicate regions of quantification: red, green, and blue boxes represent the distal, middle, and proximal neurite, respectively. The scale bar presents 10 µm. (B) Representative kymographs of indicated neurons at different regions of the neurite. (C) Quantification of EB3-mCherry comets dynamics in B. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001, one-way ANOVA followed by Dunnett’s post-hoc tests. (D) Representative kymographs of indicated neurons at different regions of the neurite. (E) Quantification of EB3-mCherry comets dynamics in D. ****, P<0.0001, two-tailed Student’s t-test. At least 15 neurons were analyzed per condition per repeat. All bar graphs are expressed as mean ± s.e.m. from three independent repeats.

HMMR regulates the localization of TPX2 on microtubules in neurons.

(A-D) Hippocampal neurons were co-transfected with indicated shRNA- and H2B-BFP-expressing plasmids at 0 DIV and cultured for 4 days before ice-cold methanol fixation. The Hmmr-targeting #1 shRNA was utilized to deplete HMMR. (A) Representative PLA images for TPX2 and β-III-tubulin (upper panel) and merged images of PLA, H2B-BFP, differential interference contrast (DIC) (lower panel) in 4 DIV hippocampal neurons. Quantification of the inter-punctal distance of PLA signals in axon (B) and dendrite (C). Only neurons possessing both PLA and H2B-BFP signals were quantified. ****, P<0.0001, two-tailed Mann-Whitney test. (D) PLA signals were presented along the neurite only when both TPX2 and β-III-tubulin antibodies were present. (E-H) Hippocampal neurons were co-transfected with H2B-BFP- and either AcGFP- or AcGFP- mHMMR-expressing plasmids at 0 DIV and cultured for 7 days before ice-cold methanol fixation. (E) Representative PLA images for TPX2 and β-III-tubulin in 7 DIV hippocampal neurons. Quantification of the inter-punctal distance of PLA signals in axon (F) and dendrite (G). Only neurons possessing both PLA and H2B-BFP signals were quantified. ****, P<0.0001, two-tailed Mann-Whitney test. All box plots are expressed as first quartile, median, and the third quartile with whiskers extending to 5-95 percentile. (H) PLA signals were presented along the neurite only when both TPX2 and β-III-tubulin antibodies were present. Scale bars represent 20 µm in (A), 5 µm in (D) (H), and 100 µm in (E).