C.P.H d

c 2_CL o D) Power P.C. <

Figure 42. Schematic o f the frequency domain FLIM system setup

(A) The excitation source was provided by the 488 nm emission line o f an Argon/Krypton gas laser. (B) The excitation light was passed through an 80 MHz resonating AOM to generate a sinusoidally modulated excitation beam. (C) Two frequency synthesizers and a high voltage amplifier accurately controlled the frequency o f modulation. The high coherent properties o f the excitation light were removed by passing the beam through a rotating ground glass disk and reconverging the scattered light with a high NA lens. The excitation light was then launched into the epi-illumination port o f the inverted microscope. (G) A dichroic mirror and emission fdter separated the excitation and emission light following sample excitation. Fluorescence light was subsequently directed onto a multichannel plate (H), the gain o f which was modulated enabling homodyne phase sensitive imaging using a CCD camera (I). An automated multi-position stage enabled the rapid high magnification imaging o f multiple cells during a single experiment (J). Peripheral devices were controlled by a Macintosh PowerPC (k). This diagram is based on a simplified version o f that previously published (Squire and Bastiaens, 1999).

3.3 RESULTS

In this study expression constructs encoding EGFP fusions o f dominant negative Cdc42,

TclO, N-WASP, and PAKl were introduced into cells using nuclear microinjection in order

to investigate the role o f their endogenous counterparts in the chemotaxis and motility o f XI5

rat sarcoma cells. Expression constructs encoding EGFP fusions o f wild-type proteins were

also used to determine whether effects seen with inhibitory proteins were the specific

consequence o f the dominant negative properties conferred by their mutations.

Much o f our understanding o f GTPase function has greatly profited from the identification,

through mutational analysis, o f functional variants that exhibit alterations in guanine

nucleotide binding and hydrolysing efficiencies (Bishop and Hall, 2000). Single amino acid

changes in the guanine nucleotide binding pocket o f Cdc42, TclO, and other GTPases have

provided dominant negative and constitutively active mutant variants o f these proteins that

have been o f great use in the study of GTPase related signalling. The introduction of the

single amino acid change T17N in Cdc42 confers a dominant negative function by locking

the protein in the GDP bound state. In this study this dominant negative variant,

Cdc42(T17N) along with a similar TclO variant TclO(T31N), were used to elucidate the role

o f the respective endogenous proteins in the chemotaxis o f rat sarcoma cells. Truncated

mutations of WASP family proteins that lack the VGA region within the carboxyl-terminus

o f the molecule have provided an effective means o f disrupting WASP-mediated signalling

to the cytoskeleton (Frischknecht et al., 1999). Deletion o f the VGA region prevents the

recruitment o f the Arp2/3 complex following protein activation, thus preventing actin

intact and thus competes with endogenous protein for activation by upstream regulators. The

N-WASP truncation mutant N-WASP(AVCA) was used in this study to disrupt the function

o f the endogenous protein. Mutations in PAKl that render the kinase domain inactive and

PAKl fragments that consist o f portions o f the autoregulatory sequence have proved

effective in inhibiting the kinase dependent signalling o f this molecule (Kiosses et al., 1999;

Zhao et al., 1998). In this study two alternative truncation mutants were used in order to

inhibit endogenous PAKl function. The first consisted of amino acids 83 - 149 of the

autoregulatory sequence, the region o f the protein responsible for masking the kinase domain

when in the autoinhibited state. The second consisted o f amino acids 1 - 1 4 9 and therefore

contained both the autoinhibitory sequence and the remaining N-terminal portion of the

protein, enabling additional membrane localization and GTPase binding.

Nuclear microinjection provided the main tool for introducing exogenous expression

constructs into cells. Dunn chamber chemotaxis assays were performed to evaluate the

directional response and motility of subconfluent cultures o f serum starved T15 rat sarcoma

cells expressing microinjected constructs. The addition o f a mixture o f PDGF-BB and IGF-1

to the outer well o f the chemotaxis chamber provided the chemotactic signal. For each

experiment a suitable field o f cells situated directly in the diffusion gap o f the chemotaxis

chamber and containing both expressing and non-expressing cells was selected for low

magnification (10 x or 20 x) observation via digital time-lapse microscopy. Sequential phase

contrast and EGFP fluorescence images were acquired at 5 min time-lapse intervals and cells

were filmed for a duration o f 16 hours. The interactive tracking o f acquired film sequences

The trajectories o f fluorescent cells from each treatment group were compared to those of

control cells expressing EGFP vector alone to assess the effects o f the various expressed

proteins on the chemotaxis, speed, and persistence o f cell movement.

Please note that while the effects o f different treatments are considered and discussed

separately in this chapter, graphically they are often presented together for ease of

comparison.

33,1 Characterisation o f T15 sarcoma cells in stable gradients o f PDGF-BB/IGF-1

A total o f 75 chemotaxis experiments were conducted to assess the role o f various polarity

proteins in the chemotaxis and motility of XI5 rat sarcoma cells. Non-expressing cells were

included in the observation field o f all recordings to provide an internal control for

chemotaxis. The data acquired from non-expressing cell populations from all 75 chemotaxis

experiments enabled a detailed analysis of the behavioural characteristics o f the T15 rat

sarcoma cell line in PDGF-BB/IGF-1 gradients. Subconfluent cultures o f T15 sarcoma cells

were previously shown to exhibit chemotaxis in mixed gradients o f PDGF-BB (60 ng/ml)

and IGF-1 (80 ng/ml) as assessed using the Dunn direct viewing chemotaxis chamber (Zicha

et al., 1991). The analysis o f cell trajectories generated from the interactive tracking o f 1959

non-expressing cells from 75 chemotaxis experiments performed in this study reconfirmed

this finding. T15 sarcoma cells exhibited a strong chemotactic response (Figure 43A,

page 19) as confirmed by the significant unimodal clustering o f cell directions towards the

source o f the chemoattractant (Rayleigh test, p < 0.001). The chemotactic response o f cells

following the commencement o f filming (Figure 43B). Cell populations also revealed a

distinctive speed profile over the course o f experiments. Cell speed typically increased over

the initial stages o f the time-course, reaching a peak after approximately 5 hours, and then

finally tailing off after a period o f approximately 8 hours (Figure 43C). XI5 sarcoma cells

exhibited a mean speed o f 14.3 pm/h in PDGF-BB/IGF-1 gradients.

Although a consistent tissue culture technique was adopted to achieve reproducible cell

densities between chemotaxis experiments, some variation between cultures was

unavoidable. Furthermore, local variations in cell density within a single culture were

difficult to control. As a consequence, variations in cell number existed between the

observation fields o f different chemotaxis experiments. Density is known to influence the

speed o f motility o f a number o f different cell types (Abercrombie, 1970). It was therefore

important to assess whether the range o f cell densities observed was sufficiently large to

significantly alter the behaviour of cell populations between experiments. This was of

importance as cell speed was one o f the aspects o f cell behaviour that was under

investigation. ANOVA revealed that no such significance existed (Figure 43D).

Consequently the range o f cell densities within which chemotaxis experiments were

A