but also by the occurrence of cytosolic crystals in ∆PEX5 mutants (sec. 10.5).
9.2 Powder Diffraction Analysis of Various Yeast
Table9.5:OverviewofsamplestestedinpowderdiffractionexperimentsatsynchrotronbeamlineP14(PETRAIII,DESY,Germany)toverifyinvivocrystallization;Yeast specificationsandtheirrespectivegrowthconditions;YPMandMMcontainedalwaysacombinationofmethanolandglycerolasdescribed;SD:synthetic-definedmedium,O: oleicacid,R:Rytka,G:glucose Nb.SpeciesStrainTypePromoter ExpressionGeneOriginMediaInc.Time [hrs]Temp.[°C] 1H.polymorphaNCYC49512DAC4T4LysozymeT4bacteriophageYPM24,36,7225,37 2H.polymorphaNCYC49512DAC4T4LysozymeT4bacteriophageMM24,3637 3H.polymorphaNCYC49512DAC4Hex-1N.crassaYPM24,48,7215,20,25,30,37 4H.polymorphaNCYC49512DAC4Hex-1N.crassaMM24,4825,37 5H.polymorphaNCYC49512DAC4Esat-6S.agalactiaeYPM24,7225,37 6H.polymorphaNCYC49512DAC4Esat-6S.agalactiaeMM2437 7H.polymorphaNCYC49512DAC4CathepsinBT.bruceiYPM24,7225,37 8H.polymorphaNCYC49512DAC4CathepsinBT.bruceiMM2437 9H.polymorphaNCYC49512DAC4PRDX5H.polymorphaYPM24,7225,37 10H.polymorphaNCYC49512DAC4EGFPA.victoriaYPM24,7225,37 11H.polymorphaNCYC49512DAC4/ ∆PEX11EGFPA.victoriaYPM2437 12S.cerevisiaeUTL7AwildtypeAOXH.polymorphaSD+O/R24,48,7230 13S.cerevisiaeBY4742wildtypeHex-1N.crassaSD+O/R24,4830 14S.cerevisiaeBY4742wildtypeHex-1N.crassaSD+G24,4830
10 | Proof of Principle Experiments for Yeast In Vivo Diffraction at XFELs
The preceding chapters delineate a conceptual approach to investigate proper-ties of the yeast peroxisomal system. Ranging from genetic engineering (ch. 8) to synchrotron-based diffraction studies (ch. 9), effort has been put into the investigation of the utilizability of H. polymorpha cells as a potential ’crystal factory’ for SFX studies at XFELs.
This chapter comprises the description of successful proof-of-principle SFX experiments with AOX nano-crystals contained in whole H. polymorpha yeast cells, at the free-electron laser LCLS (SLAC Stanford, USA). First, sections 10.1
& 10.2 show the purification of yeast peroxisomes and their characterization via DLS to estimate crystal sizes. Additionally section 10.3 presents results from a sample investigation via transmission electron microscopy, to verify AOX crystal formation and analyze sample integrity for batches of purified peroxisomes. The testing of sample injection into a X-ray beam via a liquid microjet is described in sec. 10.4. Finally diffraction studies are described in section 10.5. The presented results demonstrate the general feasibility of a yeast-based,in vivosample delivery-approach in SFX.
10.1 Purification of Mature Peroxisomes from Yeast Cells
In this section the preparation of peroxisomal fractions from two mutant yeast strains is demonstrated. Wildtype-like peroxisomal characteristics are embod-ied by one strain (WT, PMP47-GFP), whereas a PEX11 deficiency leads to different phenotype in the other mutant (∆PEX11, PMP47-GFP). Both mu-tants express the fusion protein PMP47-GFP, which is constituted of PMP47 as a peroxisome-specific adenosine triphosphate transporter and monomeric GFP. The abundance of PMP47 enables visual inspections in a microscope, but was not subjected to quantification of total fluorescence signals.
Crystal-containing yeast cells were produced according to a PAOX-induction protocol (sec. 6.2). For each cell type a total of 600 mL culture was grown in YPM media. For efficient incubation the cells were split into 3×200 mL and poured in 2 L flasks. Growth was allowed over night until early stationary phase up to an optical density of 10.
In a second step peroxisomes were purified via a two-step purification of differential- and sucrose density-centrifugation. Cells were harvested at room temperature for 5 min at3800·gand subjected to successive treatment. First protoplasting was achieved by zymolyase treatment. After disrupting the
protoplasts the total cell lysate was deployed in differential centrifugation as described, to obtain a post-nuclear supernatant (PNS). The initial differen-tial centrifugation step is thought to separate the cell organelles from the nucleus fraction and cellular debris like membranes and cell wall remains. The subsequently conducted sucrose density gradient can yield cleanly separated organelles according to their density. In a first trial the sucrose density cen-trifugation was conducted at 30000·g for 5 h. A detailed description of all methods is given in section 6.5.
To obtain a pure peroxisomal fraction a smooth linear sucrose gradient usually yields good results. This is accomplished by pouring a linear gradient in a single-use centrifugation tube from which fractions in the milliliter range can be extracted. The result is then verified with a variety of organelle-specific marker antibodies in a western blot (see sec. 8.4). As these requirements could not be fulfilled at the time of the experiments an alternative approach was developed to identify the peroxisomal fraction in layered gradients.
Instead a catalase assay based on foam formation was used for the identification of the peroxisomal fraction (sec. 6.5). The assay itself was already applied to assess catalase activities in various bacterial strains and human cells [186].
As it is known that catalase activity is a reasonable marker for peroxisomal fractions [36, 181], the foam-based assay will be exploited to optimize the purification of peroxisomes from yeast. The resulting gradient is shown in
2 3 4 5
6
7 8 1 Fraction
ΔPEX11 Wildtype Catalase Assay
30sec.
A B
1 2 3 4 5 6 7 8
C
Microscopy Image - Fraction 2
Overlay WL + GFP Fl.
Figure 10.1: (A)Sucrose-density gradient of PNS from wildtype and∆PEX11 cells after 5 hrs at 30000·g(B)Catalase activity assay conducted on fractions 1-7 (∆PEX11 cells)(C) fluorescence microscopy image, overlay of white light and FITC channel (350 ms exposure time); Scale bar: 5 µm
figure 10.1-A for both, wildtype and∆PEX11 mutant. Fractions are indicated by black lines and were chosen based on the gradient-internal density inter-faces and visible agglomerations. The density gradient is based on a protocol with sucrose concentrations of 48%, 50%, 52% and 65%, which approximately corresponds to the densities 1221, 1232, 1243 and 1319kg·m−3 [97]. Figure 10.1-B shows the results of the catalase assay for the∆PEX11 mutant, that was conducted after crude fractionation of the gradient. After an incubation time of 30 s the assay was evaluated with regards to catalase activity as indicated by the drawn red lines. For each reaction 100 µL of extracted fraction material was diluted 1:1 with homogenization buffer to decrease the overall sucrose concentration and centrifuged for 30 min at 30000·g. Thereafter the pelleted organelles were vigorously resuspended in 250µL homogenization buffer and
incubated with each 100 µL 30% H2O2 and 10% Triton X-100. The resulting foaming due to the presence of catalase was investigated. From the obtained results no distinct difference between wildtype and ∆PEX11 mutant could be found with regards to separation in the gradient. Mature peroxisomes are
Fraction
ΔPEX11 Wildtype Catalase Assay
30sec.
A B
1 2 3 4 5 6 7 8 9 10
C
Microscopy Image - Fraction 8
3 1 2
4 3
5 6 7 8 9 10
Overlay WL + GFP Fl.
Figure 10.2: (A) Sucrose-density gradient of PNS from wildtype and∆PEX11 cells after 10 hrs at 30000·g(B)Catalase activity assay conducted on fractions 1-10 (∆P11 cells)(C) fluorescence microscopy image, overlay of white light and FITC channel (350 ms exposure time); Scale bar: 5 µm
thought to have the highest density of all cell organelles in the PNS and hence were expected to accumulate at the interface between 52% and 65% sucrose solution, or eventually migrate to the bottom of the centrifugation tube [81, 104]. Interestingly, the catalase assay strongly indicated an accumulation of peroxisomes at lower densities corresponding to a sucrose concentration of 0-48% (fractions 2-4). Figure 10.1-C shows organelles from fraction 2 in an overlay of white light image and FITC channel. It clearly shows a large amount of peroxisomes apparent from the PMP47-GFP fluorescence, but unfortunately co-localized with other non-fluorescent organellar structures. Strong foaming for low density fractions is also partially explained by the presence of catalase from the cytosolic fraction and the overall protein concentration.
Furthermore the following explanations for the results can be adduced. First, mostly immature peroxisomes could populate the cells and hence end up at lower densities. A certain fraction of immature peroxisomes is always expected in a viable culture (sec. 2.2), but no visible organelles could be seen in fraction 7 or 8 when they were subjected to microscopic investigation. The latter observation would be conclusive with the second explanation, which is that the peroxisomes did not properly migrate through the gradient. Either because they stick to other cellular components or because the time of centrifugation was too short.
Hence the protocol was adapted to this new hypothesis. The time for cen-trifugation for the sucrose density gradient was increased to 10 h and the cell lysate has been treated by more extensive pipetting to achieve a stronger homogenization of the sub-cellular fractions. An inspection under the micro-scope indicated still intact organelles (data not shown). The results of the new approach are shown in figure 10.2. As expected and visible in 10.2-A the gradient appears more fractionated. Due to the appearance of a more complex purification pattern 10 layers were defined for extraction and subjected to the catalase assay (fig. 10.2-B). In both cases the same amount of protein of
about 7,5 mg·mL−1 in 5 mL was loaded onto the gradient for comparability.
To contain the foaming the total volume of reagents has been reduced by half to 50 µL each. Intriguingly it was impossible to decrease the amount of foaming reaction in lower density fractions 1-3, but it slightly increased in all higher density fractions 4-10 (see fig. 10.2-B), which is indicative for a better migration of peroxisomes. A qualitative comparison of the purifications yield a slightly increased catalase reaction at higher density fractions.
It is apparent that a lot of catalase- or catalase-like activity can be located in the low density region of the gradient. This can be due to cytosolic enzyme activities or immature peroxisomes, but might also be attributed to disrupted peroxisomes. In addition it can be assumed that a certain amount of perox-isomes gets stuck during the separation process. Prolonged centrifugtation seem to counteract on these problems by increasing the amount of catalase activity in higher density fractions, indicative for peroxisome migration.
Hereupon it was necessary to verify peroxisomal content in higher density fractions for the newly derived protocol. Therefore fraction 8-10 were pooled, centrifuged and resuspended in 100µL homogenization buffer. In a microscopic investigation of both wildtype and∆PEX11 mutant, intact peroxisomes could be verified via the PMP47-GFP marker. Figure 10.3-A and -B show examples from the pooled fractions for wildtype and ∆P11 mutant, respectively, in white light and FITC channel. Even though the findings indicate a rather pure fraction of intact peroxisomal material, many of the peroxisomes seem to cluster, which does fit to the described problems during purification. It can be assumed that the purified peroxisomes at high densities represent a subset of mature peroxisomes. Pure peroxisome fractions obtained in the same manner were subject to experimental investigation of the size distribution via dynamic light scattering (DLS) as described in section 10.2.
A B
WL GFP Fl. WL GFP Fl.
Figure 10.3: Images of purified peroxisomes (fractions 8-10) from(A)wildtype and (B)
∆PEX11 cells under white light illumination and in FITC channel (200 ms exposure time);
Scale bar: 1 µm