The non-mitotic role of HMMR in regulating the localization of TPX2 and the dynamics of microtubules in neurons
- Institute of Molecular Medicine and Bioengineering, National Yang Ming Chiao Tung University, Taiwan
- Department of Orthopedic Surgery, Changhua Christian Hospital, Taiwan
- Department of Biological Science and Technology, National Yang Ming Chiao Tung University, Taiwan
- Institute of Bioinformatics and Systems Biology, National Yang Ming Chiao Tung University, Taiwan
- Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B), National Yang Ming Chiao Tung University, Taiwan
Figures
Figure 1 with 1 supplement
Hyaluronan-mediated motility receptor (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 …
Figure 1—figure supplement 1
Overexpressing human hyaluronan-mediated motility receptor (HMMR) rescues the effect of HMMR depletion in mouse neurons.
(A) Schematic representation of shRNA targeting regions of the mouse Hmmr mRNA. The untranslated regions (UTR) are shown in white and the coding sequence (CDS) in yellow. Black bars indicate shRNA …
Figure 2 with 3 supplements
Hyaluronan-mediated motility receptor (HMMR) localizes to the microtubules in neurons.
(A) Representative images of 3 DIV mouse hippocampal neurons immunofluorescence stained with antibodies against HMMR (green) and β-III-tubulin (red). Nuclei are visualized using DAPI (blue). HMMR …
Figure 2—figure supplement 1
Validation of the hyaluronan-mediated motility receptor (HMMR) antibody.
(A) Representative images of 10 DIV mouse hippocampal neurons co-expressing the indicated Hmmr-targeting shRNA and EGFP. Neurons were immunofluorescence stained with antibodies against β-III-tubulin …
Figure 2—figure supplement 2
Transiently expressed AcGFP-mHMMR associates with microtubules in neurons.
Dissociated hippocampal neurons were transfected with AcGFP control (left) or AcGFP-mHMMR (right) on 0 DIV and fixed on 7 DIV. (A) PLA images of AcGFP control (upper left) or AcGFP-mHMMR (upper …
Figure 2—figure supplement 3
Hyaluronan-mediated motility receptor (HMMR) does not colocalize with microtubule plus-ends in neurons.
(A) Representative images of a 3 DIV hippocampal neuron immunofluorescence stained with antibodies against HMMR and EB1. The white box in the upper right panel indicates the magnified region shown …
Figure 3
Hyaluronan-mediated motility receptor (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 …
Figure 4
Hyaluronan-mediated motility receptor (HMMR) regulates the dynamics of neuronal microtubules.
(A) Representative image of a 4 DIV EB3-mCherry-expressing cortical neuron. The color boxes indicate regions of quantification: red, green, and blue boxes represent the distal, middle, and proximal …
Figure 5
Hyaluronan-mediated motility receptor (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 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (M. musculus) | Hmmr | GenBank | Gene ID: 15366 | |
| Transfected construct (M. musculus) | shRNA #1 | RNAi Consortium shRNA Library via RNAi Core of Academia Sinica | TRCN0000311803 | Lentiviral construct to express the Hmmr-targeting shRNA |
| Transfected construct (M. musculus) | shRNA #2 | RNAi Consortium shRNA Library via RNAi Core of Academia Sinica | TRCN0000311805 | Lentiviral construct to express the Hmmr-targeting shRNA |
| Transfected construct (M. musculus) | shRNA #3 | RNAi Consortium shRNA Library via RNAi Core of Academia Sinica | TRCN0000071592 | Lentiviral construct to express the Hmmr-targeting shRNA |
| Antibody | Anti-acetylated-α-tubulin (mouse monoclonal) | Abcam | ab24610 | IF (1:1000) |
| Antibody | Anti-β-III-tubulin (TUJ1) (mouse monoclonal) | BioLegend | 801202 | IF (1:4000) |
| Antibody | Anti-β-III-tubulin (TUBB3) (rabbit polyclonal) | BioLegend | 802001 | IF (1:2000) |
| Antibody | Anti-neurofilament (SMI312) (rabbit polyclonal) | BioLegend | 837904 | IF (1:1000) |
| Antibody | Anti-GFP (mouse monoclonal) | DSHB | 12A6 | IF (1:100) |
| Antibody | Anti-HMMR (E-19) (goat polyclonal) | Santa Cruz Biotechnology | sc-16170 | IF (1:50) |
| Antibody | Anti-MAP2 (rabbit polyclonal) | MilliporeSigma | AB5622 | IF (1:1000) |
| Antibody | Anti-TPX2 (rabbit polyclonal) | Oliver Gruss; Gruss et al., 2002 | IF (1:2000) | |
| Recombinant DNA reagent | pCAG-AcGFP-mHMMR (plasmid) | This paper | AcGFP-mHmmr expression vector | |
| Recombinant DNA reagent | pEGFP-hHMMR (plasmid) | Christopher Maxwell; Maxwell et al., 2003 | ||
| Commercial assay or kit | Duolink proximity ligation assay | Sigma-Aldrich | DUO92101 | |
| Software, algorithm | Prism | GraphPad v8.4.3 | RRID: SCR_002798 | |
| Software, algorithm | Fiji | Fiji | RRID: SCR_002285 | |
| Software, algorithm | NIS-Elements | Nikon | RRID: SCR_014329 |
