The non-mitotic role of HMMR in regulating the localization of TPX2 and the dynamics of microtubules in neurons

A functional nervous system is built upon the proper morphogenesis of neurons to establish the intricate connection between them. The microtubule cytoskeleton is known to play various essential roles in this morphogenetic process. While many microtubule-associated proteins (MAPs) have been demonstrated to participate in neuronal morphogenesis, the function of many more remains to be determined. This study focuses on a MAP called HMMR in mice, which was originally identified as a hyaluronan binding protein and later found to possess microtubule and centrosome binding capacity. HMMR exhibits high abundance on neuronal microtubules and altering the level of HMMR significantly affects the morphology of neurons. Instead of confining to the centrosome(s) like cells in mitosis, HMMR localizes to microtubules along axons and dendrites. Furthermore, transiently expressing HMMR enhances the stability of neuronal microtubules and increases the formation frequency of growing microtubules along the neurites. HMMR regulates the microtubule localization of a non-centrosomal microtubule nucleator TPX2 along the neurite, offering an explanation for how HMMR contributes to the promotion of growing microtubules. This study sheds light on how progenitor cells utilize proteins involved in mitosis for non-mitotic functions.

Methods
Indirect immunofluorescence staining

Cells on coverslips were fixed with 3.7% formaldehyde for 15 min at 37 °C and then washed three times with PBS. Fixed cells were permeabilized with 0.25% triton X-100 in PBS for 5 min at room temperature or extracted in -20 °C methanol for 10 min. For experiments that required cytosolic pre-extraction, cells on coverslips were permeabilized in 0.1% triton X-100 in PIPES buffer (0.1 M PIPES pH 6.9, 1 mM MgCl2, and 1 mM EGTA) for 15 sec, washed once with PIPES buffer, and fixed with 3.7% formaldehyde in PIPES buffer at 37 °C for 30 min and then washed with PBS three times. Cells were then blocked with 10% BSA in PBS for 30 min at 37 °C, incubated for 1 hour at 37 °C with different primary antibodies: GFP (1:100), HMMR (1:50), MAP2 (1:1000), SMI312 (1:1000), TPX2 (1:2000), acetylated-α-tubulin (1:1000), TUBB3 (1:2000), and TUJ1 (1:4000). After primary antibody incubation, cells were washed with PBS three times and incubated with AlexaFluor-conjugated secondary antibodies (1:1000). All antibodies were diluted in 2% BSA in PBS. Coverslips with cells were washed with PBS three times and mounted with Fluoromount onto glass slides.

In situ proximity ligation assay (PLA)

Cells were fixed in -20 °C methanol for 10 min, washed with PBS, and then blocked in a chamber with Duolink II Blocking Solution for 30 min at 37 °C. Primary antibodies used for different experiments were diluted in PBS containing 2% BSA at aforementioned dilutions and incubated for 1 h at 37 °C. Cells were then incubated with PLA probes diluted in Antibody Diluent for 1 h at 37 °C. Subsequent procedures were conducted according to the manufacturer’s instructions.

Microscopy acquisition

Fluorescence images were acquired on a Nikon Eclipse-Ti inverted microscope equipped with a Photometrics CoolSNAP HQ2 CCD camera, an Intensilight epi-fluorescence light source, and Nikon NIS-Element imaging software. 20 × 0.75  N.A. or 60 × 1.49 N.A. Plan Apochromat objective lenses were used to collect fluorescence images.

Live cell imaging was performed on a Nikon Eclipse-Ti inverted microscope equipped with a TIRF illuminator and a Tokai Hit TIZHB live cell chamber. Images were acquired using a 60 × 1.49 N.A. Plan Apochromat objective lens, a 561 nm DPSS laser, a Photometrics CoolSNAP HQ2 camera, and Nikon NIS-Elements imaging software. The built-in perfect focus system (PFS) was activated to maintain axial position. Images were acquired every 500 milliseconds over a 2-minute period. Only the neurons with clear EB3 comets were imaged.

Image analysis

For neurite length analysis, fluorescence images were manually traced with the ImageJ plugin NeuronJ 1.4.1. Only neurons expressing both the transfection indicator (e.g., EGFP) and specific markers (e.g., β-III-tubulin, MAP2, SMI312) were analyzed. Only neurites longer than its soma diameter were analyzed.

For axon branching analysis, neurites were manually traced using the Fiji plug-in Simple Neurite Tracer (Longair et al., 2011) before being processed with the Fiji plug-in Sholl analysis.

For acetylated microtubule quantification, manually generated linescans along the neurite were used to obtain the signal of acetylated-α-tubulin and β-III-tubulin. The ratio of acetylated-α-tubulin/β-III-tubulin was calculated to represent the level of microtubule acetylation.

For microtubule plus-end dynamics analysis, NIS-Elements software was used to generate the kymograph for the EB3-mCherry images. All kymographs were generated using a window 10 μm in length and 7 pixels in width. For proximal neurite analysis, the kymograph window started from the edge of the soma and extended outwards. For mid-neurite analysis, the kymograph window was centered at the midpoint of the neurite. For distal neurite analysis, the kymograph window started at the wrist of the growth cone and extended inwards. The speed and persistence time of EB3-mCherry were quantified from the kymograph by drawing a line along an EB3-mCherry event. Only EB3-mCherry movements that could be followed clearly for equal or more than four frames (1.5 seconds) were defined as an event. The emanating frequency of EB3-mCherry was quantified from the kymograph by counting the number of EB3-mCherry events per minute.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 8. Significant differences between the means were calculated with the indicated statistical methods.