Metas de Producto, línea de base y Meta esperada para el cuatrienio:

Live cell imaging of rerouting kinetics was performed using an Olympus IX71 in heated glass-bottom dishes at 37ºC (Bioptechs Delta T5 µ-environmental culture dish controller) in CO2-independent medium (Invitrogen) supplemented with 10% FBS and 100 units/ml penicillin/streptomycin, using Cell-R acquisition software and a Hamamatsu ORCA-ER C4742-80 camera with a 60× oil-immersion objective (1.42 NA). Quantification of GFP intensity during KS was performed using ImageJ/Fiji, with an ROI that defined the spindle and one that excluded it, and plotted as ΔF/F0. The GFP-FKBP rerouting movie was acquired by Stephen Royle, who also performed the curve-fitting of the kinetics data for all conditions.

3.3.5.2

Immunofluorescent labeling

HeLa cells on 16 mm diameter glass coverslips were fixed with PTEMF (50 mM PIPES [1,4-Piperazinediethanesulfonic acid], 10 mM EGTA, 1 mM MgCl2, 0.2% Triton X-100, 4% paraformaldehyde, pH 7.2) at room temperature for 15 min, or methanol at -20ºC for ch-TOG staining. Cells were then permeabilised using PBS with 0.5% Triton X-100 for 10 min and blocked for 1 hour using PBS with 5% BSA and 5% goat serum. The following antibodies (diluted in blocking solution) were used: (1) mouse monoclonals: clathrin heavy chain (X22, CRL-2228 ATCC; 1 in 2000), TACC3 (ab56595, Abcam; 1 in 1000), and Eg5 (611186, BD Biosciences; 1 in 500) (2) mouse polyclonal: GTSE1 (H00051512-B01P, Abnova; 1 in 100), (3) rabbit polyclonals: ch-TOG (34032, Autogen Bioclear; 1 in 1000) and β-tubulin (ab6046, Abcam; 1 in 2000), NuMA (3888s, Cell Signaling; 1 in 1000), pericentrin (ab4448, Abcam, 1 in 1000), HURP (kind gift from Erich A. Nigg, University of Basel, Switzerland; 1 in 1000). Fluorescently conjugated secondary antibodies were Alexa488, Alexa568 or Alexa633 (Molecular Probes; all used at 1 in 500). Coverslips were mounted on microscope slides using Mowiol containing 4’,6- diamidino-2-phenylindole (DAPI).

3.3.5.3

Epifluorescence microscopy

Epifluorescent micrographs were taken using a Nikon Eclipse Ti-U microscope with standard filter sets for visualisation of DAPI, GFP, mCherry/Alexa Fluor 568 and Alexa Fluor 633, a Nikon Digital Sight DS-Qi1Mc camera, a 60× (1.40 NA) oil- immersion objective and NIS acquisition software.

3.3.5.4

Confocal microscopy

For TACC3 quantitation experiments, identical laser power and acquisition settings were used. Fluorescence intensity quantification was performed using ImageJ/Fiji by measuring mean pixel density in a 20×20 pixel ROI placed in the spindle region (determined using the tubulin immunolabel channel in the same cells) and background subtracted.

3.4

Results

3.4.1

The timescale and kinetics of protein rerouting by KS during

mitosis

Live cell imaging of TACC3 KS was performed in HeLa cells at metaphase (Figure 3.3A). The removal of TACC3 from the spindle was fast, taking around 5 min (Figure 3.3A, C), and this was similar in clathrin rerouting experiments (Figure 3.3B). These results indicate that TACC3 is dynamic, cycling on and off MTs with quite a high frequency despite being in a complex with ch-TOG and clathrin.

To analyse the parameters of protein removal using KS, live cell imaging was performed on HeLa cells expressing mCherry-MitoTrap and GFP-FKBP-TACC3 or GFP-FKBP in interphase or mitosis and treated with 200 nM rapamycin. Rerouting of GFP-FKBP was extremely fast in both interphase and mitosis with almost identical kinetics (t1/2= 3.1 and 4.2 sec, respectively; Figure 3.4). Interestingly, the rerouting of GFP-FKBP-TACC3 was slower than GFP-FKBP, and rerouting of GFP- FKBP-TACC3 was considerably faster during interphase compared to

Figure 3.3: Knocksideways and timescale of spindle protein removal during mitosis. A. Representative video still images of a TACC3-depleted HeLa cell expressing GFP-FKBP-TACC3 and mCherry-MitoTrap. Rapamycin (200 nM) was added at t0. GFP-FKBP-TACC3 is depleted from the spindle and co-localises with mCherry-MitoTrap. B. Representative video still images of a HeLa cell expressing GFP-FKBP-LCa (green) and mCherry-MitoTrap (red). Rapamycin (200 nM) was added at t0, and GFP-FKBP-LCa is rerouted to mCherry-MitoTrap. C. Quantification of GFP ∆F/F0 in the indicated areas over time during TACC3 KS in A.

Figure 3.4: Comparing kinetics of protein rerouting by knocksideways. A. Graph of rerouting kinetics for GFP-FKBP-TACC3 (blue) or GFP-FKBP (green) to mitochondria in interphase (lighter colour) or mitosis (darker colour), single cell examples. Rapamycin (200 nM) was applied in each case (indicated by the arrowhead). An overlay of curve fits to describe the rerouting are shown on the same time scale. B. Description of kinetics. GFP-FKBP-TACC3 in mitosis was best fit by the Hill logistic function, all other were best fit by a double exponential function.

mitosis (t1/2=11.0 and 243.6 sec, respectively; Figure 3.4). It is likely that these differences reflect the variable affinity of the target proteins for cellular substrates. Indeed, GFP is situated throughout the cell and is not known to bind any endogenous proteins in either interphase or mitosis, which may explain the very fast and similar rerouting kinetics observed in both interphase and mitosis.

TACC3 is not bound to MTs during interphase, therefore it seems likely that GFP- FKBP-TACC3 would be free in the cytosol during this stage of the cell cycle, explaining its fast rerouting to mitochondria. The considerable slowing of TACC3 rerouting during mitosis (t1/2=243.6 sec) compared to interphase (t1/2=11 sec) seems therefore likely to be due to MT-binding during mitosis, and dependent on the rate at which TACC3 cycles on and off the spindle.

How can the slower TACC3 rerouting in interphase compared to GFP be explained? Inducing the expression of GFP-TACC3 in interphase causes TACC3 to form aggregates in the cytosol. Although these aggregates disassemble when rapamycin is added (not shown), the slower rerouting kinetics compared to GFP-FKBP may be explained by the presence of the aggregates themselves, which could slow the release of GFP-FKBP-TACC3 located in the centres of the aggregates. Another explanation would be the difference in size between GFP (26.8kDa (Prasher et al., 1992)); and GFP-TACC3 (26.8 + 90 kDa; (Still et al., 1999)). It has previously been found that protein diffusion in the cytoplasm is a decreasing function of protein size (Arrio- Dupont et al., 2000).

3.4.2

Efficient protein inactivation by KS is dependent upon the