Investigating Akt signalling mechanism using single-molecule

imaging technique in living cells

Yue Wan Lin

University of Tokyo Research Internship Program (UTRIP) 2016

Ozawa Laboratory, Department of Chemistry, School of Science, The University of Tokyo, Japan

ABSTRACT

Many studies have established that the PI3K/Akt pathway is involved in numerous cellular

processes, such as cell growth, survival and metabolism. However, the detailed mechanism

of how the pathway functions, including the activation of Akt and its subsequent activity, is

still unclear. In this project, we used single-molecule TIRF imaging to observe the

localisation of mutant Akt molecules on the plasma membrane of HeLa cells before and after

stimulation. Wild type Akt and a dominant negative mutant (K179M) of Akt, which shows no

activity, was compared with two other mutants, T308A and S473A, to study the contribution

of these phosphorylation sites on Akt to its role in the pathway. T308 is found to be more

important than S473 in Akt activation and functionality, while S473 is more important for

membrane localisation. This novel method of observing Akt has provided critical insight into

how the phosphorylation sites on Akt contribute to its role in the PI3K/Akt pathway.

1. INTRODUCTION

1.1 Akt / Protein kinase B (PKB) and signal transduction

Akt, or protein kinase B (PKB), is a serine/threonine protein kinase that has a key role in the

phosphoinositide-3 kinase (PI3K)/Akt pathway (Lasserre et al., 2008). Akt consists of three

domains: a pleckstrin homology (PH) domain at the amino terminal, followed by a kinase

domain, and a regulatory domain at the carboxyl terminal (Figure 1). The PH domain is

involved in recognition of PIP3, and phosphorylation sites (threonine-308 and serine-473) are

found in the kinase and regulatory domains respectively (Song, Ouyang, & Bao, 2005).

Figure 1: Akt protein structure. A PH domain is present at the amino terminal, followed by a kinase domain (containing K179 and T308), and a regulatory domain (containing S473) at the carboxyl terminal. T308 and S473 are phosphorylation sites on Akt (Song, Ouyang, & Bao, 2005). When K179 is mutated to methionine, Akt kinase activity is lost (Franke et al., 1995)

The PI3K/Akt pathway is crucial in generating many cellular responses upon stimulation by

extracellular factors, including cellular growth, survival and glucose metabolism (Figure 2).

After binding of specific factors (e.g. growth factor, insulin), receptor tyrosine kinases (RTKs)

are activated and in turn activate phosphatidylinositol 3-kinase (PI3K) by phosphorylation.

PI3K then adds a phosphate group to phosphatidylinositol-4,5-bisphosphate (PIP2) to

produce phosphatidylinositol-3,4,5-triphosphate (PIP3) on the plasma membrane. Both Akt

and phosphoinositide-dependent kinase-1 (PDK1) attach to PIP3, and Akt is phosphorylated

by PDK1 at threonine-308 (T308). Additionally, Akt is phosphorylated at serine-473 (S473)

by mammalian target of rapamycin complex 2 (mTORC2), which is also activated by the

RTK signalling pathway. Activated Akt then phosphorylates a variety of downstream targets,

such as caspase-9, Tuberin and glycogen synthase kinase 3, to generate many

physiological effects (Lasserre et al., 2008; Manning & Cantley, 2007).

Figure 2: A simplified illustration of the PI3K/Akt pathway (Yoshimura, 2015). 1) Receptors are first activated by binding to specific ligands. 2) Active receptors directly or indirectly activate PI3K. 3) Activated PI3K phosphorylates PIP2 on the plasma membrane to produce PIP3. 4) Akt from the cytosol binds to PIP3, and subsequently gets activated via phosphorylation by other kinases. Active

PH Kinase Regulator

y

T308 S473 K17

9

Akt

Akt then phosphorylates other downstream targets to generate a variety of cellular responses specific to the initial stimuli (Manning & Cantley, 2007).

Despite the large variety of substrates targeted by Akt, cellular responses are specific to the

stimuli that initiate the PI3K/Akt pathway. Thus, Akt is likely to selectively activate certain

substrates in response to different stimuli. However, the mechanisms through which this

occurs is still unclear, and various studies have proposed different methods through which

the selectivity can be achieved (Manning & Cantley, 2007).

1.2 Single molecule imaging and total internal reflection fluorescence (TIRF)

Single molecule imaging allows for observations of individual particles in live cells with high

accuracy of spatial and temporal localisations. This information enables quantitative analysis

of how the molecules behave and interact, which can help to explain their functions. In

addition to tracing the positions and motion of individual molecules, single molecule imaging

can also track their activation through fluorescence resonance energy transfer (FRET). For

example, interaction with downstream targets can be tracked to detect the activation as

activation is commonly followed by such interactions. Molecules on the plasma membrane

can also be imaged via total internal reflection fluorescence (TIRF) microscopy, as shown in

Figure 3. Lowered rates of diffusion in the plasma membrane would allow the molecules to

be traced with less difficulty (Kusumi et al., 2014).

Figure 3: TIRF microscopy. When the angle of incident light at the interface is at least equal to the critical angle, the incident light undergoes total internal reflection and an evanescent wave is produced in the medium. The wave intensity reduces exponentially as distance from the interface increases, limiting the penetration of light into the medium. Only fluorophores within the restricted distance are excited such that no out of focus fluorescence is detected (Newcastle University, 2011).

1.3 Investigation aims

This study aims to further investigate the mechanism of Akt activation and activity by

analysing the contribution of the phosphorylation sites of Akt to its localisation on the plasma

membrane. Point mutations were introduced at the phosphorylation sites of Akt, creating two

Akt mutants – T308A and S473A. Single molecule TIRF microscopy and analysis were

conducted and the results compared positive and negative controls. Wild type (WT) Akt

serves as the positive control and K179M, an Akt mutant which has no kinase activity

(Franke et al., 1995) serves as a negative control. The number of the different Akt molecules

on the plasma membrane of HeLa cells was analysed to quantify the effect of the mutations

on Akt activity.

2. MATERIALS AND METHODS

2.1 Plasmid construction

A gene encoding the fusion protein of Akt1 linked to a C-terminus HaloTag (Promega Corp.)

was previously generated and inserted into pcDNA4/v5-HisB plasmid to create the template

plasmid. PrimeSTAR GXL DNA polymerase (Takara Bio Inc.) was then used for PCR to

introduce point mutations into the Akt gene in the template. Different forward and reverse

primers were used to generate the mutant plasmids K179M, T308A and S473A. T308 and

S473 were mutated to alanine because its methyl group side chain is small and unreactive.

The non-mutated template plasmids were removed via treatment with DpnI (Takara Bio Inc.)

at 37°C for 2 hours. The plasmids were then purified with EtOH and EDTA, and eluted with

Milli-Q water. Competent DH5α E. coli cells (Takara Bio Inc.) were then transformed with the

mutant plasmids via heat shock at 42°C. The E. coli were then plated on LB plates

containing ampicillin to select for successful transformants and incubated for 16 hours at

37°C. Three colonies were then picked from each plate and grown in 3ml of LB medium

containing ampicillin for 16 hours at 37°C.

Plasmids were then extracted from E. coli and purified using FastGene Plasmid Mini Kit

(Nippon Genetics Co. Ltd.) and sent for sequencing (Eurofins Genomics) to ensure that the

correct sequences were obtained. The fusion gene constructs encoding mutant Akt-Halo

were then cut out through double digestion with XbaI and KpnI (Takara Bio Inc.), followed by

gel electrophoresis (0.8% agarose, EtBr) to isolate the desired gene sequences of 2367bp

each. New pcDNA4/v5-HisB plasmids were also digested with XbaI and KpnI, followed by

SAP (Takara Bio Inc.) treatment to prevent self-ligation. Mutant Akt-Halo fusion genes were

then reinserted into new pcDNA4/v5-HisB plasmids using solution I from DNA ligation kit

(Takara Bio Inc.) to ensure that there are no mutations in the rest of the plasmid sequence.

2.2 Plasmid isolation and amplification

The recombinant plasmids were then used to transform competent DH5α E. coli cells via

heat shock before incubation for 16 hours at 37°C. Colony PCR was conducted on six

colonies from each plate using GoTaq Green Master Mix (Promega Corp.), and forward and

reverse primers targeting sequences on the insert and vector respectively. Gel

electrophoresis (0.8% agarose, EtBr) was then used to check for recombinant plasmids that

have been amplified. Two colonies from each plate with the recombinant plasmid were then

picked and incubated in 3ml of LB medium with ampicillin for 16 hours at 37°C.

The plasmids were then extracted from E. coli and purified using FastGene Plasmid Mini Kit

(Nippon Genetics Co. Ltd.) and sent for sequencing (Eurofins Genomics) to ensure that the

correct sequences were obtained. After identifying plasmids containing the correct

sequence, the corresponding colonies were picked and used for large scale incubation in

80ml of LB with 1% ampicillin for 16 hours at 37°C. High purity plasmids were then extracted

using PureLink HiPure Plasmid Midiprep Kit (Invitrogen) and used for transfection.

2.3 Cell culture and transfection

HeLa cells were cultured in DMEM high glucose medium with L-glutamine and phenol red

(Wako Pure Chemical Industries, Ltd.), supplemented with 10% FBS (Sigma-Aldrich Co.)

and 1% penicillin-streptomycin (Gibco). The cells were seeded onto 35mm glass-bottom

dishes at a density of 3 x 105 cells per dish, and incubated at 37°C with 5% CO2 for six

hours. Serum starvation was then carried out by incubating the cells in DMEM high glucose

medium with L-glutamine and phenol red (Wako Pure Chemical Industries, Ltd.) for 18 hours

before transfection.

Transfection was then carried out with 3µg of plasmids containing one of the four different

Akt genes – WT, K179M, T308A and S473A. OptiMEM (Gibco), Lipofectamine 3000 and

P3000 reagent (Invitrogen) were used for the transfection. The cells were then incubated for

four hours at 37°C with 5% CO2 before observation using single-molecule TIRF microscopy.

2.4 Single molecule imaging and analysis

HaloTag TMR ligand (Promega Corp.) was used to label Akt-Halo fusion proteins expressed

by the cells. Excess TMR was washed off using HBSS (Gibco). Laser light of 561nm was

used for excitation. A 100x NA 1.49 oil immersion TIRF objective (Olympus Corp.) was used

in conjunction with AquaCosmos software (Hamamatsu Photonics) to obtain videos of 10

seconds each showing the Akt-Halo motion on the plasma membrane. 2µl of 50ng/ml

platelet-derived growth factor (PDGF) was then added to each sample and the cells were

observed after two, three and five minutes. The videos were converted to.avi format using

Fiji (ImageJ) software and then analysed using WinATR (Kusumi Lab) software to quantify

the number of single molecules.

3. RESULTS

3.1 Before PDGF stimulation

Four cells each were analysed for WT, K179M and T308A, and three cells for S473A. First,

the average number of Akt-Halo on the plasma membrane before PDGF stimulation was

compared (Figure 4). All three mutants were observed to have more Akt-Halo on the

membrane than WT before stimulation.

Figure 4: Graph showing the average number of Akt-Halo localised on the plasma membrane of HeLa cells transfected with the four types of plasmids before PDGF stimulation. WT shows the lowest Akt-Halo membrane localisation and T308A shows the highest localisation. Error bars indicate standard deviation from the calculated average value.

This result could be due to all three mutations preventing Akt-Halo activation such that, after

localisation to the membrane, Akt-Halo cannot leave the membrane for downstream activity.

Of the three mutants, T308A has the most Akt-Halo on the membrane before stimulation,

which indicates the highest extent of Akt-Halo activation inhibition. This suggests that T308

is the most important site for Akt activation, which is consistent with the finding that T308 is

located in the kinase domain activation loop of Akt (Fayard, Tintignac, Baudry, & Hemmings,

2005).

3.2 Before and after PDGF stimulation

Next, the data was normalized for comparison across cells transfected with different

plasmids before and after PDGF stimulation (Figure 5). The number of Akt-Halo on the

membrane in each cell in each condition was normalised to that of the corresponding cell

before stimulation. However, very large standard deviations were observed for most of the

data, making the average values unsuitable for studying the effect of the mutations.

Figure 5: Graph showing the normalised average number of Akt-Halo on the plasma membrane before and after stimulation across cells transfected with the four types of plasmids. Number of Akt-Halo after stimulation was normalised to that before stimulation in the same cell before averaging. Error bars indicate standard deviation from the calculated average value.

The data was then divided into three categories according to the observed trend after

stimulation for better comparison: increase, decrease, and constant. A change of ±0.3 from

the unstimulated condition was arbitrarily set as the threshold for categorisation as

increasing or decreasing trends.

3.2.1 Increasing trend

The increasing trend was observed in four cells, each expressing one type of Akt-Halo

(Figure 6). WT was observed to have the largest increase in number of Akt-Halo on the

membrane after PDGF stimulation, suggesting that WT is the most functional Akt-Halo

molecule. The smallest increase was observed in T308A Akt-Halo, suggesting that it has the

least functionality. This could mean that T308 is the most important site for Akt function out

of the three mutated sites.

Figure 6: Graph showing the normalised number of Akt-Halo on the plasma membrane for cells showing an increasing trend after PDGF stimulation. Number of Akt-Halo after stimulation was normalised to that before stimulation in the same cell before averaging. Error bars indicate standard deviation from the calculated average value.

3.2.2 Decreasing trend

The decreasing trend was only observed in cells transfected with the mutants: one K179M,

two T308A and one S473A (Figure 7). It is possible that the mutant Akt-Halo molecules have

a lower binding affinity to PIP3 than endogenous Akt, and are thus displaced by endogenous

Akt upon stimulation with PDGF. The largest decrease is observed in S473A Akt-Halo,

suggesting that it has the lowest binding affinity to PIP3. S473 could thus be the most

important out of the three sites for PIP3 binding. With WT Akt-Halo, the binding affinity is

likely to be similar to endogenous Akt. In addition, there are less Akt-Halo on the membrane

before stimulation for WT than any of the other mutants (Figure 4). These factors could

explain the lack of a decreasing trend in cells expressing WT Akt-Halo.

Figure 7: Graph showing the normalised number of Akt-Halo on the plasma membrane for cells showing a decreasing trend after PDGF stimulation. Number of Akt-Halo after stimulation was normalised to that before stimulation in the same cell before averaging. Error bars indicate standard deviation from the calculated average value for conditions with data from more than one cell.

Cells showing a decrease in mutant Akt-Halo also have more mutant Akt-Halo on the plasma

membrane prior to stimulation than other cells of the same mutant (Figure 8). This

oversaturation of Akt-Halo on the membrane could lead to competition between endogenous

and mutant Akt-Halo for binding to PIP3 binding upon PDGF stimulation. Less mutant Akt-

Halo would thus be observed on the plasma membrane after PDGF stimulation if it has lower

affinity for PIP3 binding than endogenous Akt, consistent with the previous hypothesis.

Figure 8: Graph showing the number of mutant Akt-Halo on the plasma membrane for each cell. Red, blue and grey lines indicate the cells showing a decreasing, increasing and constant trend after stimulation respectively.

Cells expressing mutant Akt-Halo also show an increasing trend when initial number of

mutant Akt-Halo on the membrane is lower. This suggests that the number of Akt-Halo on

the membrane prior to stimulation is a factor contributing to the different responses of

observed cells to PDGF stimulation.

3.2.4 Constant

No change was observed before and after stimulation in at least one cell expressing each

type of Akt-Halo: three WT, two K179M, one T308A, one S473A (Figure 9). This Indicates

that not all cells were responsive to PDGF stimulation, or that not all cells respond to the

same extent to the stimulation.

Figure 9: Graph showing the normalised number of Akt-Halo on the plasma membrane for cells showing no change before and after PDGF stimulation. Number of Akt-Halo after stimulation was normalised to that before stimulation in the same cell before averaging. Error bars indicate standard deviation from the calculated average value

4. DISCUSSION

Large standard deviations were present in the data due to the limited number of samples

obtained for analysis. The small sample size is insufficient to fully study the effect of the

mutations as it is unable to reflect the behaviour of cell populations as a whole. Different

responses of cells to PDGF stimulation led to further deviation such as due to different

expression levels of Akt-Halo and different sensitivity to PDGF stimulation. These

differences could cause large deviations on the results due to the small sample size.

Single molecule imaging also suffers from high signal to noise ratio (SNR), especially due to

the low expression levels required (Kusumi et al., 2014). This could lead to many false

positives and negatives in the results obtained. Another issue in single molecule imaging is

photobleaching (Kusumi et al., 2014), and it has been reported that photobleaching of TMR

occurs after ~1.5s (Mossuto et al., 2014). This implies that photobleaching would have a

significant impact on the results from this investigation, decreasing the number of molecules

detected. This would be especially evident in the observation at five minutes after stimulation

since multiple observations of ~10s each were made for each cell.

It is also possible that the plasmids introduced were unstably expressed in the cells as

observations revealed the presence of several overexpressed cells as well as cells with no

Akt-Halo within the same sample. All of these factors could have contributed to a lowered

accuracy of the results, and need to be taken into consideration when looking at the results.

5. FUTURE DIRECTIONS

First, it is necessary to make observations of more cells to get more conclusive and

reproducible data. Cells of similar expression levels could be chosen for analysis and

comparison, such as through establishing a stable cell line. SNR also needs to be improved

through selection of an appropriate expression level of Akt-Halo. Shorter or less

observations could also be considered to prevent photobleaching from affecting the results.

Transfection with a lower amount of plasmids and a longer incubation after transfection

could also improve accuracy by stabilising the plasmid expression in transfected cells.

In addition, knockdown or knockout of endogenous Akt in cells prior to transfection could

also be considered to prevent overexpression of Akt in the cells. Downstream studies could

also be carried out to see the effects of Akt mutations on cellular response produced by the

PI3K/Akt pathway.

Finally, observation of endogenous WT and mutant Akt-Halo directly could also be

performed, such as through trans-splicing or using the CRISPR-Cas9 system. The low

expression levels of Akt-Halo required for single molecule imaging must be taken into

consideration when designing such experiments.

6. CONCLUSION

In conclusion, this investigation has found that individual cells can have very varied

responses to PDGF stimulation, such as due to different initial conditions present in the cells.

Analysis of the limited data obtained seems to suggest that both phosphorylation sites of Akt

have significant contributions to its role, with T308 being more important for activation and

function of Akt, and S473 being more important for localisation of Akt to the membrane.

However, more data is necessary before the validity of these conclusions can be verified.

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