- Transform the generated Rrm4-GFP expression plasmid into
U. maydisas described in Subheading3.2 [32–34]. - Inoculate yeast-like growing cells in 3 mL of CM-glc in a glass
tube and grow culture on rotation wheel for 24 h at 28C. - Inoculate a main-culture in a 250 mL baffled flask by mixing
15 μL of the 24 h culture with 30 mL CM-glc medium and
incubate it for 15–20 h shaking with 200 rpm at 28C. - For induction of hyphal growth measure the optical density
(OD 600 ) of the cultures and centrifuge the corresponding vol-
ume at 3500gfor 5 min to obtain an OD 600 of 0.5 in 20 mL.
Wash once with NM-glc media and resuspend the cells in 20 mL
of the same media to induce hyphal growth (seeNote 3).
Continue shaking with 200 rpm at 28C for 7–8 h. - Prepare agarose cushions by pipetting 250μL melted agarose
solution on a microscope slide. Add a second slide directly on
top of the agarose drop, parallel to the lower slide. After
20–30 min one slide can be removed by carefully sliding it
sideways. - Pipet 1μL of the cell culture onto the cushion, distribute the
cell culture by swaying the slide and let it dry for 1–2 min (see
Note 4). - To visualize motile Rrm4-GFP signals choose appropriate illu-
mination for the excitation of chosen fluorophores (here GFP,
488 nm excitation); use the 63or the 100objective and
apply a laser power (100 mW) of 30–40%. - Record movies with 150 frames and 150 ms exposure time at
each frame (seeNote 5). - Use the camera in stream mode to visualize movement
throughout the hyphae and ensure the fastest acquisition.
Rrm4 shuttles bidirectionally along microtubules throughout
the whole cell (Fig.2a). - After recording moving Rrm4-Gfp signals, convert the movie
into a kymograph, which plots traveled distance over time
using the software package MetaMorph (Fig.2a and b;see
Note 6). Processively moving Rrm4-Gfp signals are depicted
as diagonal lines (Fig.2a and b). - The velocity of moving RBPs can be measured by marking such
processive Rrm4-Gfp signals. For this, the start and end point
of the moving signal is connected by a line (Fig.2b). The
travelled distance per time represents the velocity of the
moving signal (Software package Metamorph, Version 7).
326 Sabrina Zander et al.