Single molecule tracking of Tom40p in live yeast mitochondria reveals firm anchoring of the protein transport machinery
Stoyan Tankov
Single molecule tracking of Tom40p in live yeast mitochondria reveals firm anchoring of the protein
transport machinery
StoyanTankov
Sammanfattning
Tom40 är ett integralt protein i det mitokondriella yttermembranet som fungerar som en kanal för proteinimport in i mitokondrier. Här studerar vi dynamiken av jäst‐Tom40 fuserat med det fotoomvandlingsbara fluorescenta proteinet Dendra2b genom att använda en kombination av stroboskopisk laserexcitation med stokastisk fotoaktivering och superupplösningsfluorescensavbildning.
Vi visar att Tom40 uppvisar ett extremfall av instängd diffusion, att det är hårt fäst i membranet och ”gungar” med en amplitud av ~0.3 µm på submillisekundtidskala.
Det här uppförandet antyder att proteintransportmaskineriet i mitokondrier är årt fäst i membranet, snarare än fritt diffunderande som tidigare antagits.
h
Examensarbete 30 hp Biologi B programmet Uppsala University July 2010
Abbreviations
AOM Acousto‐optical modulator DPSS Diode‐pumped solid‐state
nce contrast
DIC Differential interfere
EMCCD Electron‐multiplying charge‐coupled device
roscopy
FP Fluorescent protein
M ed localization mic
photobleaching
FPAL Fluorescence photoactivat FRAP Fluorescence recovery after
otein
GFP Green fluorescent pr
MSD Mean square displacement
NA Numerical aperture
PALM Photoactivated localization microscopy M
SPT Single particle tracking
STOR Stochastic optical reconstruction microscopy TOM Translocase of the outer membrane
IM Translocase of the inner membrane IRF Total internal reflection fluorescence T
T
C
ontents:
1 Abstract... 3
2 In oduction ... 4tr 2.1 Mitochondria: function and structure... 4
2.2 Mitochondrial protein transport system ... 5
2.3 Dynamics of the mitochondrial membrane proteins... 6
2.4 Optical microscopy ... 6
2.5 Labels for SPT investigations... 8
2.6 Single Particle Tracking ... 9
3 Aims of the project...11
4 Materials and Methods...12
4.1 The optical setup...12
4.2 Construction of the Tom40_Dendra2b fusion...13
4.3 Sample preparation and data collection ...14
4.4 Single‐molecule tracking and analysis of experimental trajectories ...15
5 Results...16
5.1 SPT of Tom40_Dendra2b...16
5.2 SPT of immobilized Dendra2b ...17
6 Discussion and outlook...19
7 Conclusions...20
8 Acknowledgments...21
9 References ...22
1 Abstract
Tom40 is integral protein of the mitochondrial outer membrane that functions as a channel for protein import into the mitochondria. Here, we study the dynamics of yeast Tom40 fused with the photo‐convertible fluorescent protein Dendra2b using a combination of stroboscopic laser excitation with stochastic photoactivation and super‐resolution fluorescence imaging.
We demonstrate that Tom40 exhibits an extreme case of confined diffusion, being anchored in the membrane firmly and “wobbling” with amplitude of ~0.3 µm on the sub‐millisecond timescale. This behavior suggests that the protein transport machinery in the mitochondria is firmly anchored in the membrane, rather than diffusing freely as was postulated before.
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2 I troduction
2.1 Mitochondria: function and structure
Mitochondria are essential organelles of almost all eukaryotic cells and take part in many cellular processes such as energy production, apoptosis and fatty acid metabolism to name a few. They contain a functional genome, which is considered to be a relic from the times when their ancestors were free‐living organisms.
Mitochondria have two membranes (inner and outer), which divide them into two compartments: intermembrane space and matrix (Figure 1).The Inner membrane contains the power plant of the mitochondia – the ATP synthase particles. The ATP synthase machinery converts the H+ gradient between the matrix and the intermembrane space into ATP. The Inner membrane has a complex geometry, folding into cristae, which increases its area considerably. The matrix contains mitochondrial DNA and ribosomes, as well as so‐called granules, which are a storage form of metal ions, and play a role in the regulation of ionic balance in the organelle.
Figure 1. Schematic structure of a mitochondria (figure courtesy of wikipedia.
rg).
o
The mitochondrial genome codes for only 8 proteins (Foury et al., 1998) whereas proteomic studies have revealed that the total number of yeast mitochondrial proteins is about 800 (Reinders et al., 2006). Thus, the majority of the mitochondrial proteins have to be imported from the cytosol, passing through the two membranes. This constitutes a non‐trivial engineering problem, and a specialized system of transport was developed to that end.
2.2 Mitochondrial protein transport system
The process of protein import into mitochondria from the cytosol is driven by a multi‐component system (for review, see (Chacinska et al., 2009)). These components are located in the cytosol, in both mitochondrial membranes, in the intermembrane space and in the mitochondrial matrix. The process occurs in everal steps and protein components of both the outer and inner membranes are nvolved (Fig. 2).
s i
Figure 2. Schematic structure of mitochondrial protein transport ystem (adopted from Rehling at s
al., 2004).
First, cytosolic proteins are recognized by the receptors of the TOM‐
complex (Translocase of the Outer Membrane) and are then transported through the outer membrane via the channel protein Tom40 (for review, see (Perry et al., 2008)).
The integral proteins of the outer membrane are inserted into it by another outer membrane complex, SAM (Sorting and Assembly Machinery) (for review, see (Walther &
Rapaport, 2009)). Proteins targeted to the other mitochondrial sub‐
compartments undergo further transport through the inner membrane. Depending on their final destination, transported proteins are recognized by one of the two ifferent inner membrane translocases: TIM22 and TIM23 (for review, see (Bauer d
et al., 2000) and (Jensen & Dunn, 2002)).
Tom40 is the integral protein of the outer mitochondrial membrane and it is a central component of the TOM‐complex. It belongs to the class of β‐barrel membrane proteins and forms pores with a diameter of about 22 – 25 Å (Hill et al.,
1998, Schwartz & Matouschek, 1999). Due to the oligomerization of several individual Tom40 molecules (Ahting et al., 2001) and interactions with other omponents the architecture of the pore formed in organello can be very intricate for review, see (Perry et al., 2008)).
c (
.3 Dynamics of the mitochondrial membrane proteins 2
Little is known about the dynamics of membrane proteins in the mitochondria, and most of the data has been produced using the FRAP (Fluorescence Recovery After Photobleaching) technique applied to mitoplasts, rather than on live mitochondria.
FRAP is almost exclusively done on cytochrome c, a central component of the electron transfer chain (Vanderkooi et al., 1985, Hochman et al., 1982), and information regarding the dynamics of the components of the outer membrane such as Tom40 is lacking altogether. Electron microscopy data (van der Klei et al., 1994, Schulke et al., 1997) show no obvious patterns in Tim and Tom distribution, which was interpreted as an evidence of their free diffusion, as no direct observations of this diffusion were ever made.
O
2.4 ptical microscopy
2.4.1 Principles of optic microscopy
In order to determine the distribution and dynamics of the TOM complex, one needs to use an appropriate technique, which would produce data with temporal and spatial resolution comparable with the scale on which TOM complexes exert their functions (nms and µs). Also, techniques should preferably be non‐invasive, since interference with the dynamic nature of the TOM complex could result in un‐
natural structures.
Light microscopy is a non‐invasive technique allowing investigations of the structures using light diffraction on the sample, and it can (at least in certain variants) fit to the temporal and spatial resolution criteria outlined above. The main components of the light (or optical) microscope are:
1) Light source. In the simplest set ups light from the sun is used, but in the modern scientific microscopes dedicated light sources such as halogen lamps or lasers are used. The main advantage of these is more
homogeneous illumination, both in terms of intensity and (especially in the case of lasers) in terms of pectral haracteristics. s c
2) Condensor.The condensor focuses the light from the light source on the sample, where it diffracts.
3) Objective. The objective collects the light diffracted from the sample. The higher is the ability of the objective to collect light, the higher is the potential of the microscope to resolve the structure of the sample.
Numerical Aperture (NA) is used as a measure of this resolving power. NA equals index of refraction of the media in which the light travels multiplied on the sine of the half‐ angle of the maximum cone of light the objective can collect (NA = n·sinθ).
NA is directly linked to maximum resolution of the setup (d): d = λ/2NA, where λ is the wavelength of the imaging light.
4) Ocular or eyepiece.The eye pieces serves to produce the image on the retina of the eye from the light passed on by the objective. The magnification powers of the eye piece and the objective multiply, thus giving the final magnification of the microscope.
5) Frame. Usually all the abovementioned components are assembled on the frame which ensures the proper relative orientation and protects the microscope from stray light which does not come from the illuminated sample.
2.4.2 Fluorescent microcopy
Optical microscopes can operate in a variety of modes, but the one which is of special interest for us, is fluorescence microscopy. In this technique fluorescent labels are used, and these emit light with a specific wavelength. In combination with the appropriate spectral filters (so called dichroic filters), which allow only light of a particular wavelength to pass, these labels provide us with the ability to observe only the fluorescent molecules.
This approach is especially powerful when fluorescent molecules are sparse enough and the optical system is powerful enough so that single fluorescent molecules can be detected and their evolution in time can be analyzed. This way we can observe a whole array of transport phenomena, such as diffusion, active transport, etc, as well as reconstruct the structures with a sub‐difractional resolution. This approach when single molecules are observed and their movements are recorded, is dubbed Single Particle Tracking (SPT).
Some fluorescent molecules can be turned on and off upon illumination with a photo‐activating beam. These fluorofors are called photoconvertible (when the switch from one fluorescent state to another is irreversible) or photoswitchable (if fluorescent can be turned on and off with light of specific wavelengths). The positions of spawned individual molecules can be determined by Gaussian fit, and then all individual positions are used to piece together a super‐resolution structure of the object studied. This approach allows breaking the diffraction limit, since
i u e
there is no need to resolve ind vid al molecules from each oth r.
Several different variants of this approache are in used; fluorescence photoactivated localization microscopy (FPALM)(Hess et al., 2006), photoactivated localization microscopy (PALM)(Betzig et al., 2006) and stochastic optical
n st et al., 2006).
reco struction microscopy (STORM)(Ru 2.5 Labels for SPT investigations
SPT allows tracking with high precision the diffusion of individual microscopic particles attached to relevant molecules. The information garnered from measurement of particle trajectories provides useful information about the mechanisms and forces that drive and constrain the motion of particles.
This approach requires specialized labels to be attached to the biological molecules of interest. These probes should have certain characteristics(Fernandez‐Suarez &
Ting, 2008) as described below.
First, they should have narrow emission spectra, so that their signal can be easily separated from the signal coming from other parts of the sample. Second, they should be bright ‐ they should emit a lot of photons with a well‐defined wavelength when illuminated with the imaging light. Third, they should be stable under in vivo conditions. Forth, they should be easily and specifically linked to the molecule of
ld not alter the behavior of the target considerably.
choice and they shou 2.5.1 Organic dyes
Organic dyes are small and bright, and they have a very good spectral characteristics (Fernandez‐Suarez & Ting, 2008). However, they have a major drawback: attaching them to the molecule of choice is a very tricky problem. If the molecule of interest is produced inside the cell (e.g. a protein) than attaching the dye co‐translationally is almost impossible. A modified protein has to be micro‐
injected in the cell, which makes in vivo investigations very challenging and almost
s uch as bacteria or mitochondria.
impos ible in small objects s 2.5.2 Fluorescent proteins
Fluorescent proteins (FP), such as GFP variants, are rather poor fluorophores.
They are bulky, not very bright and are very much affected by the environment (Bogdanov et al., 2009a, Bogdanov et al., 2009b). However, using recombinant DNA technology it is relatively easy to create fusion proteins consisting of the protein of interest and the FP.
One such protein is Dendra2b(Gurskaya et al., 2006), which was used in our study.
ch can be converted by the UV light.
This is a photoconvertable FP whi
S
2.6 ingle Particle Tracking 2.6.1 General principles of SPT
SPT basically allows observation of single molecules. However, single molecules are subject to diffusion. In so‐called normal diffusion, which is random, and unbiased Brownian motion, Mean Square Displacement (MSD) is proportional to the diffusion constant (D) and time (t): MSD = 2·d·D·t (here d is dimensionality of the system, which is 2 for plane dimension and 3 for three dimensional systems).
However, in biological systems particles usually cannot diffuse freely. First, they are limited in space by the cell dimensions (Deich et al., 2004), thus MSD curves have a plateau. Second, they can be actively transported, such as MreB bacterial tubulin(Kim et al., 2006). Numerous other deviations from the Brownian diffusion are also possible.
Precision in single molecule localization is proportional to the square root of the amount of the photons collected from the fluorophore (Thompson et al., 2002):<(Δx2)>= s2 / N, where Δx is precision of the particle localization, s is the standard error of the fitting function (usually Gaussian) and N is the number of photons collected.
2.6.2 Use of SPT for investigations of membrane proteins
SPT techniques have revolutionized investigations of protein diffusion in membranes. However, SPT is almost exclusively applied to eukaryotic cell membrane proteins, demonstrating that they exhibit confined diffusion: proteins
freely diffuse in microdomains (corrals) of the membrane (Douglass & Vale, 2005, Vrljic et al., 2002). These corrals are produced by the underlying cytoskeleton Douglass & Vale, 2005) and are relatively big, with a characteristic size of 2 µm, (
which is comparable with the size of whole bacterial cell or mitochondria.
Much less SPT data is available for bacterial membrane proteins. One relatively well studied protein is the maltoporin LamB, which was shown to exhibit confined diffusion (Gibbs et al., 2004), though the temporal resolution of the study (1 per second frame rate) provided little information on the fast dynamics of the system.
These results are consistent with LamB data obtained using optical tweezers, which also suggested confinement, attributed to LamB attachment to the ptoreoglican between the 2 membranes (Oddershede et al., 2002). Another bacterial membrane protein which was studied by SPT, PelC, was tracked with a imilar temporal resolution (0.9 s), and it exhibited very slow normal Brownian s
diffusion (D = 12±2 10‐3 µm2/s) (Deich et al., 2004).
Mitochondrial membrane proteins have never been investigated using SPT, thus we can only guess about their diffusion properties. Mitochondria do not have a cytoskeleton, or proteoglycan layer between the outer and inner membranes, thus one could expect Tom40 to undergo free diffusion in the outer membrane, as has previously been suggested (van der Klei et al., 1994, Schulke et al., 1997).
3 Aims of the project
Yeast mitochondria are a very promising model system for in vivo single molecule investigations since yeast is highly genetically amenable, readily producible, and, as we established during this study, yeast mitochondria have a low background of fluorescence. Therefore we set to investigate the mitochondrial protein transport machinery using SPT in live mitochondria.
The primary aim of the project was to develop a proof‐of‐principle using a Dendra2b‐Tom40 fusion. Investigation of Tom40 dynamics will lay the groundwork for later investigations of the protein transport machinery. Labeling of other components of the mitochondrial membrane would also pave the way to multicolour studies of mitochondrial dynamics, complemented with single molecule in vivo FRET investigations of protein and tRNA transport.
thods 4 Materials and Me
4.1 The optical setup
A schematic diagram of the optical setup is shown in Figure 3. Below is given a brief overview.
An acousto‐optical modulator (AOM, IntraAction, 40 MHz) shutters a wide‐field yellow excitation laser beam into an Olympus IX81 inverted microscope. The excitation laser light from the 555‐nm diode‐pumped solid‐state (DPSS) laser (CrystaLaser) is first filtered (Z550/20x, Chroma Technology) and then focused onto the back aperture of an Olympus total internal reflection fluorescence (TIRF) objective ( ). The objective collimates the light and excites a 4‐µm wide area at the sample plane with a laser power of 50 to 200 .
Motorized stage
Long-pass dichroic
Long-pass dichroic AOM
Flip- lens T132 shutter
NA 1.45 objective
EMCCD camera
Band-pass filter Photo-
conversion
Fluorescence- excitation
Figure 3. Schematic diagram of the optical setup.(based on English et al,2010)
The AOM is synchronized with a PhotonMAX electron‐multiplying charge‐coupled device (EMCCD) camera (Princeton Instruments) by a NI‐DAQ M‐series data acquisition card (PCI‐6259, National Instruments), and is controlled via LabVIEW
8.5 (National Instruments) to pass short 0.4‐ or 1‐ms excitation pulses in the
id n
m dle of each imagi g frame.
A photoconversion laser beam at 405 nm (Radius, Coherent) is spatially overlapped using a long‐pass dichroic filter (Z405RDC, Chroma Technology) and is focused at the sample plane. An anamorphic prism pair (GPA‐3X‐8.0‐405, CVI MellesGriot) and a 50‐µm pinhole (Thorlabs) correct the asymmetry of this laser beam to produce a TEM00 mode. The laser is independently shuttered using a UNIBLITZ T132 shutter controller that delivers 3‐ms photoconversion pulses at regular time intervals at powers ranging from 0.1 to
A long‐pass dichroic filter (Z555RDC, Chroma Technology) is used to separate excitation and photoconversion laser light from the fluorescence emission of Dendra2. The emission light is further filtered using a band‐pass emission filter (HQ605/75m, Chroma Technology).
4.2 Construction of the Tom40_Dendra2b fusion
Construction and validation of theTom40_Dendra2b fusion were performed in the aboratory of Dr. PiotrKamenski, Moscow State University by Anton Kuzmenko,and l
thus are not discussed in great detail.
For the construction of the Tom40_Dendra2b fusion, we used the yeast strain YKB14–1a [tom40::HIS4, his4519, leu23 112, Δura3, ade2, YEplac42R (TOM40::URA3)] (Gabriel et al., 2003), kindly provided by Trevor Lithgow (University of Melbourne, Australia). The Tom40 C‐terminus was chosen for attachment of Dendra2b because the N‐terminus contains signals for itochondrial transport. Addition of Dendra2b to the N‐terminus would be xpecte
m
e d to disrupt Tom40 integration into the membrane.
TOM40 gene was first cloned into the pRS415 shuttle vector containing LEU3 as a marker gene using NotI and XhoI sites. Then the site for NcoI restriction endonuclease was created in the resulting plasmid directly after the stopcodon of TOM40 gene using the QuikChange mutagenesis kit (Stratagene). Finally, the Dendra2b coding sequence was inserted into this plasmid using yeast recombination in vivo. For this, the plasmid containing the NcoI site was linearized with NcoI enzyme. In parallel, Dendra2b coding sequence was amplified by PCR from the pDendra2 plasmid (a gift from D. Chudakov, Institute of Bioorganic Chemistry, Moscow, Russia). The primers for this PCR contained 40‐nt long 3’‐
proximal parts homologous to the corresponding ends of the linearized plasmid.
Then, the W303 yeast strain was transformed simultaneously by these two DNAs using the lithium acetate / PEG method (Gietz & Woods, 2006). Yeast cells containing the product of homologous recombination between two linear DNA
fragments were selected on medium without leucine. The resulting plasmid coding for the Tom40_Dendra2b fusion protein was isolated using the QiaPrep plasmid purification kit (Qiagen) (Singh & Weil, 2002) and, using the plasmid shuffling approach (Rose, 1990) used to replace YEplac42R plasmid in the YKB14–1a strain esulting in a yeast strain expressing the fusion protein instead of the wild‐type r
Tom40p.
The mitochondrial respiration of the mutant yeast strain was assayed in vivo by direct measurement of the oxygen uptake using the Clark electrode and polarographOxygraph (Hansatech Instruments) at 30°C. W303 and YKB14–1a strains were used as the controls. Growth of the yeast cells on the medium containing non‐fermentable carbon source (glycerol) served as an alternative method for assaying mitochondrial function. Serial 10X dilutions of the mutant yeast strain culture with initial OD600 = 0.1 were plated on this medium, together with the same dilutions of the control strains W303 and YKB14–1a.
In both experiments strains contacting Tom40_Dendra2b and wt Tom40 showed o significant difference, thus validating the Tom40_Dendra2b fusion construct.
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4.3 Sample preparation and data collection
The yeast mitochondria were isolated according to (Entelis et al., 2002) with minor modifications. Yeast cells were cultivated in 200 mL of selective media (Bio101 Inc) to OD600 of 2‐3 at 30°C, harvested by centrifugation and disrupted with glass beads (Sigma, d = 0.5 mm). After two rounds of differential centrifugation (4000 g nd 15000 g), mitochondria were loaded on a two‐step sucrose gradient (0.6 M / a
1.85 M).
Thereafter, obtained mitochondria were immediately subjected to microscopy.
Mitochondria were immobilized in coverslips coated with poly‐L‐lysine and detected using the Differential Interference Contrast (DIC) mode of the microscope (Figure 4). Coating was formed by repetitive washing of the coverslips with 0.02%
poly‐L‐lysine and deionized w ter. A series of dilutions were prepared in order to determine mitochondrial concentration optimal for imaging. a
Figure 4. DIC image of mitochondria with an overlay of the fluorescence signal from Tom40_Dendra2b (in red).
Immobilized mitochondria were imaged for 20 000
The time‐lapse movies were manually curated and frames with photoconversion pulses or with no single molecule traces were removed. Example frames are shown in Figure 5.
e
Figur 5. Example frames of the Tom40_Dendra2b fluorescent trace at 10 Hz frame rate.
4.4 Single‐molecule tracking and analysis of experimental trajectories racking procedures were performed as described elsewhere (English et al., T
2010).
Single Tom40 fluorophores were tracked in hand‐edited movies using the particle tracking software Diatrack (v3.03, Semasopht), which identifies and fits the intensity spots of our fluorescent particles with symmetric 2D Gaussian functions.
All routines for trajectory analyses are written in IGOR Pro 6.12A by ArashSanamard and Dr. Brian English (Dr. J. Elf’s laboratory, Uppsala University).
The single‐molecule diffusion trajectories are analyzed by calculating MSDs(Deich et al., 2004, Niu & Yu, 2008) for all possible time intervals in the sample (x‐y) plane.
5 Results
5.1 SPT of Tom40_Dendra2b
Mitochondria were isolated according to a published procedure (Entelis et al., 2002) with minor modifications, attached to poly‐L‐lysine coated coverslips and imaged (see Materials and Methods). We used a strobing approach, i.e. using the same exposition time (5 ms) we varied the frame time (5, 20, 100, 500 and 1000 ms)(English et al., 2010). This approach allowed us to track Tom40 in the same photophysical conditions in a wide time range, spanning from ms to seconds, observing both fast and slow processes.
Figure 6.Single‐molecule Tom40_Dendra2b tracking in live S. cerevisiae mitochondria. A. Overlay of 7 single‐molecule Tom40_Dendra2b trajectories in S. cerevisiaelive mitochondria with a frame time of 5 ms and an exposure time of 5 ms. B. Mean Square Displacements (MSDs) analysis for a frame time of 5 ms and an exposure time of 5 ms. C. MSDs analysis for a frame time of 100 ms and an exposure time of 5 ms. D. MSDs analysis for a frame time of 1000 ms and an exposure time of 5 ms.
The results were quite surprising. Already at 5 ms time intervals Mean Square Displacement (MSD) plots reached the plateau, suggesting that Tom40 was confined in a ~0.3 µm micro‐corral (Figure,6A,B). Importantly, Tom40 was effectively sampling this space within the time below 5 ms. Imagine at different frame rates, spanning from 5 ms to 1 sec (Figure 6, BCD) the MSD did not change its character, suggesting that even at the 1 second time scale Tom40 was firmly anchored in the mitochondrial membrane.
Tom40 showed confinement within a ~0.3 µm micro‐corral and yet its fast dynamics within that space could have an alternative explanation: Tom40 could be absolutely immobile, and the observed MSD plateau could be the result of the short
ignal itself).
noise (noise associated with the photon s 5.2 SPT of immobilized Dendra2b
As a control for instrument drift we first imaged Tom40 with a coverslip glued to the optical stage, which did not change the results (results not shown). Secondly we imaged Dendra2b molecules immobilized on the poly‐L‐lysine slides (Figure 7A‐D). Here we also observed MSD, which did not increase with time, but the plateau was much lower.
Figure 7.Dendra2b tracking. A. Single‐molecule Dendra2b trajectory with a frame time of 5 ms and an exposure time of 5 ms. B. Mean Square Displacements (MSDs) analysis for a frame time of 5 ms and an exposure time of 5 ms. C. MSDs analysis for a frame time of 20 ms and an exposure time of 5
s. D. MSDs analysis for a frame time of 100 ms and an exposure time of 5 ms.
m
The efficiency of GFP‐based fluorophores is greatly affected by the environment (Bogdanov et al., 2009b), thus this could be a concern for comparison of Tom40 which is localized in the mitochondrial intermembrane space and free Dendra2b in the buffer solution. Precision in localization is proportional to the square root of the amount of the photons collected from the fluorophore(Thompson et al., 2002), thus differences in the MSD plateaus could simply reflect the differences in the short noise. Therefore photon count analysis was needed in order to conclusively demonstrate the nature of the differences in the MSD plateaus of stuck Dendra2b and Tom40_Dendra2b.
6 Discussion and outlook
Our results suggest that Tom40 diffusion is profoundly different from that observed with eukaryotic membrane proteins, it diffuses relatively freely but within domains (Douglass & Vale, 2005), similarly to the diffusion pattern of bacterial membrane protein PelC(Deich et al., 2004). The closest example is bacterial protein maltoporinLamB, which showed a similar extreme confinement (Oddershede et al., 2002, Gibbs et al., 2004). However, there is a profound difference between our SPT tracking data and that presented in (Gibbs et al., 2004): our analysis has much greater time resolution, milliseconds vs seconds.
It is tempting to hypothesize on the nature of this extraordinary behavior of Tom40. In the case of LamB, confinement was attributed to interactions with intermembane proteoglycans(Oddershede et al., 2002). In the case of Tom40 no intermembrane proteoglycan matrix is present: thus it has to interact with something else. One possible culprit could be a rivet‐like interaction formed by TIM and TOM connecting smooth outer membrane and highly creased inner membrane (Schulke et al., 1999, Schulke et al., 1997), connecting outer and inner membranes.
Yeast mitochondria are a very promising model system for in vivo single molecule investigations since yeast are highly genetically amenable, readily produced, their mitochondria have low background fluorescence, and they are devoid of efflux pumps present in bacteria. Thus the delivery of small molecules to the inside is not experimentally challenging. Labeling of other components of the mitochondrial membrane would pave the way to multicolour studies of mitochondrial membrane dynamics, complimented with single molecule in vivo FRET investigations of protein and tRNA transport.
7 Conclusions
1) We have constructed a yeast strain encoding a fusion of the Tom40 mitochondrial transport channel and the GFP variant Dendra2b, and demonstrated the feasibility of single molecule tracking in live mitochondria. This paves the way to constructing and tracking other similarly labeled components of the mitochondrial protein transport machinery, which in the future can produce a dynamic description of this system with unprecedented temporal and spatial resolution.
2) The Tom40 protein transport channel does not exhibit free diffusion in the mitochondrial membrane. It is virtually immobile on a timescale spanning from ms to seconds, suggesting that protein transport complexes are fixed in the outer mitochondrial membrane.
8 Acknowledgments
I am grateful to all the people who participated in the project. First, I am grateful to Anton Kuzmenko who constructed the Tom40_Dendra2b fusion and taught me how to purify and handle yeast mitochondria. Second, I am grateful to Dr. Brian English, who taught me the basics of microscopy and introduced me to SPT techniques. Third, I am grateful to Arash Sanamard, who shared the Igor scripts for the SPT analysis. And, lastly, I am grateful to my supervisors, Dr Vasili Hauryliuk and Dr Johan Elf, who participated at all the stages of this project.
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