Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

Podsumowanie

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Streszczenie

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Wprowadzenie

Microtubules (MTs) are highly dynamic structures found in virtually all eukaryotic cells and in some bacteria¹. Together with actin and intermediate filaments, they sculpt the cytoskeleton^2,3. Cell division⁴, molecule transport⁵, flagellar beating⁶, the sensation of the surrounding environment through primary cilium⁷, hearing (kinocilium)^8,9, embryogenesis^10,11,

Access restricted. Please log in or start a trial to view this content.

Protokół

1. Seeding of HeLa Cells

1. Prepare 2 mL of 5 μg/mL fibronectin solution in phosphate buffered saline (PBS) and add 450 μL of it into each well of a 4 well chambered coverslip (#1.5). Incubate the slide for 15 min at 37 °C and 5% CO₂.
2. Rinse asynchronously growing HeLa cells with Dulbecco’s Phosphate Buffered Saline (DPBS) and incubate with trypsin-EDTA (0.05%: 0.02%; w:v) for 5 min at 37 °C. Stop the enzymatic reaction by the addition of Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) at 3:1 (v:v) ratio of added trypsin-EDTA.
   NOTE:

Access restricted. Please log in or start a trial to view this content.

Wyniki

Following the given protocol outlined in Figure 1A, the pEB3-tdTomato plasmid was transiently expressed in asynchronously growing HeLa cells. The cells were synchronized 48 h after the transfection at prometaphase through DME treatment (Figure 1B). This step ensured that the measurement of MT dynamics was always done at the same phase of the cell cycle. The time-lapse movies were further processed and analyzed with U-Track v2.2.0 as described in its supplementar.......

Access restricted. Please log in or start a trial to view this content.

Dyskusje

Here, we describe a modification of a method first established by Ertych et al.⁴⁴. Along with several other modifications, we combine this technique of MT dynamics analysis with dual spinning disk confocal imaging. The use of the dual spinning disk improves the resolution of growing MTs while reducing phototoxicity³⁶. We further reduce the photobleaching and laser light-induced damage of the cells by switching to a longer wavelength fluorescent reporter. The tdTomato fluore.......

Access restricted. Please log in or start a trial to view this content.

Materiały

Name	Company	Catalog Number	Comments
Dimethylenastron	Merck	324622
DMEM w/o phenol red	Gibco	31053-28
DPBS	Gibco	14190-094
Fetal bovine serum	Biochrom	S0415
Fibronectin Bovine Plasma	Merck	F4759	Sterile powder
GlutaMAX	Gibco	35050-038	Stable glutamine substitutive
jetPRIME	Polyplus	114-15
EB3-TdTomato	Addgene	plasmid #50708
RPMI 1640	Gibco	61870-010
Trypan Blue	Merck	T8154-20ML
Trypsin/EDTA solution	Biochrom	L2143	0.05%/0.02 % w/o calcium and magnesium
µ-slide	Ibidi	80426	4-well slide with #1.5 coverslip
Eclipse Ti Inverted microscope	Nikon	NA
Objective	Nikon	MRD01991	CFI Apo TIRF 100xC Oil
ACAL Laser Excahnger	Nikon		Laser box. 405, 458, 488, 514, 561 and 647 nm
Spinning disk module	Andor	CSU-W
Camera	Andor	iXon Ultra 888
Environmental Chamber	Okolab		Dark chamber equipped with CO2 supply, temperature control and humidifier
HeLa Cells	DSMZ	ACC-57
NIS Elements v4	Nikon		Spinning disk microscope. Acquisition Software
MATLAB	Mathworks		Computing environment
Prism 8	GraphPad		Statistical analysis and display software