Excluded-Volume Effects in Living Cells**

Biomolecules evolve and function in densely crowded and highly heterogeneous cellular environments. Such conditions are often mimicked in the test tube by the addition of artificial macromolecular crowding agents. Still, it is unclear if such cosolutes indeed reflect the physicochemical properties of the cellular environment as the in-cell crowding effect has not yet been quantified. We have developed a macromolecular crowding sensor based on a FRET-labeled polymer to probe the macromolecular crowding effect inside single living cells. Surprisingly, we find that excluded-volume effects, although observed in the presence of artificial crowding agents, do not lead to a compression of the sensor in the cell. The average conformation of the sensor is similar to that in aqueous buffer solution and cell lysate. However, the in-cell crowding effect is distributed heterogeneously and changes significantly upon cell stress. We present a tool to systematically study the in-cell crowding effect as a modulator of biomolecular reactions.


S.1.1 PEG FRET sensor
Polyethylene glycol (PEG) with a molecular weight of 10 kDa was synthesized containing amino terminal modifications. Atto488 and Atto565 were conjugated to both ends of the PEG chain. The synthesized PEG sensor was obtained by Rapp Polymere (Tübingen, Germany).
Prior to use, PEG sensor was dissolved in DPBS at pH 7.4 (Sigma-Aldrich) in the desired concentrations.

S.1.2 Oocyte extract preparation
Freshly prepared oocytes were kindly provided by the lab of M. Hollmann (Department of Biochemistry I, Ruhr-University Bochum). Isolated oocytes were collagenase treated for 1 h at 20 °C in Ca 2+ -free Barth's saline to remove the follicle cell layer. The enzymatic reaction was stopped by washing with Ca 2+ -Barth's saline. Healthy oocytes were selected and used for extract preparation. The extraction was performed similar as described elsewhere. [1] Briefly, oocytes were washed several times with extraction buffer (50 mM sucrose, 100 mM Kcl, 0.1 mM CaCl, 10 mM HEPES-KOH pH 7.7). Cells were transferred to a 2 ml reaction tube and carefully sedimented at 1000 xg. Surplus buffer was removed. 200 µl Nyosil M25 was added on top and then centrifuged at 1000 xg for 1 min allowing for tighter packing of the oocytes. Supernatant oil and buffer was removed and the cells lysed for 15 min at 20,000 xg and 2°C. Translucent yellow cytoplasm was then transferred into new reaction tubes and a 100x protease inhibitor cocktail (Sigma-Aldrich) was added. The mixture was centrifuged at 20,000 xg and 2 °C for 15 min. Again, cytoplasmic fraction was recovered and the protein concentration was determined using Bradford assay with BSA as standard.

S.1.5 Measurement procedure
In vitro and in-cell measurements were performed using the same experimental setup: Measurements were performed at room temperature with fast alternating excitation using a For the measurements, either a 63x/1.2 oil immersion objective (Zeiss) or a 40x/0.95 air objective was used (Zeiss). Excitation light was directed on the probe using a DFT 490+575 (HE) beam splitter (Zeiss). Emission light was guided to a FT 565 (HE) beam splitter (Zeiss) to separate donor and acceptor emission. Donor emission was passed through a BP 512/30 HE filter (Zeiss) and acceptor emission was passed through a BP 630/98 HE filter (Zeiss). Images from donor and acceptor emission were recorded simultaneously using two AxioCam HS (Zeiss) cameras and the AxioVision 4.8.2 (Zeiss) software.

S.1.6 Data analysis
For in-cell data, FRET efficiency was calculated as described by Feige et al. using the PixFRET plug-in for ImageJ (NIH). [2] In vitro data was evaluated using a self-written Matlab code. For all measurements, background data (either from non-injected cells for cell measurements or from buffer without sensor for in vitro measurements) was subtracted separately from each channel. The FRET efficiency was calculated by: [3] = !"#$ denotes the intensity of Atto565 with 470 nm excitation, !"#"$ the intensity of the donor with 470 nm excitation, !""#$%&' the Atto565 intensity with 555 nm excitation, !"#"$ the correction factor for Atto488 emission bleed into the Att565 channel and !""#$%&' the correction of direct excitation of Atto565 by 470 nm excitation. Statistical analysis was performed using Graphpad Prism.

S.1.3 In vitro measurements
Different crowding solutions (Ficoll70, sucrose, TMAO, PEG 10 kDa, BSA; all Sigma-Aldrich) were prepared at different concentrations in DPBS, pH=7.4 (Sigma-Aldrich) and mixed with the PEG-FRET sensor to yield a final concentrations of 5 µM. For the DNA measurements, ssDNA from Salmon testis (Sigma-Aldrich) was used. The solution was placed on glass bottom dishes (fluorodish, WPI) and the measurements were performed with an Axio Observer Z1 (Zeiss) inverse microscope as described before.

S.1.4 Cell culture
HeLa cells were grown in standard T25 culture flasks (Sarstedt) in DMEM media (Sigma-Aldrich) supplemented with 1% penicillin-streptomycin (PS) and 10% fetal bovine serum (FBS) in a humidified atmosphere at 5% CO2 and 37°C. Cells were sub cultured every 2 days after reaching approximately 80-90 % confluence using standard trypsin digestion and split in a 1:4 to 1:5 ratio into freshly prepared T25 flasks. For experiments, HeLa cells were plated 1 day prior to injection at a density of 2 x 10 5 cells on a 35 mm glass bottom dish.

S.1.5 Microinjection
Microinjection was used to deliver the sensor into HeLa cells using standard protocols. [4] Briefly, HeLa cells were injected 1-2 d after plating. Therefore, the culture media was aseptically removed and exchanged with Leibovitz L15 (Sigma-Aldrich) supplemented with 30 % FBS. Injection was performed using an Eppendorf FemtoJet connected to an Eppendorf InjectMan NI2. FemtoTips II were filled from the back side with a 2 mg ml -1 stock solution of sensor and connected to the FemtoJet. The injection parameters were adjusted for each injection so that approximately 5 % of the cell volume was injected. As a starting point, an injection pressure of 100-150 hPa, a compensation pressure of 35 hPa, which causes a constant efflux from the capillary and prevents dilution of the sensor, and an injection time of 0.5 s was used. A few tens of cells were injected within a timeframe of 10 -15 min. The injected cells were incubated for 10 min at RT to minimize background fluorescence caused from leakage of the sensor from the capillary. Viable cells were imaged within 30 min after injection. Microinjected cells were considered viable when they showed nuclear integrity, adherence to the glass bottom as well as a constant morphology, as reported earlier as criteria for cellular health. [4a,4b,5] We further showed that cells were viable by nuclear staining using the DNA dye Hoechst 34580 (Sigma-Aldrich). We showed that no changes in adherence, morphology or nuclear integrity were observed before and after injection ( Figure S7a). Cells remained viable even 3 h after injection ( Figure S7b). Further, nuclear injection did not influence cell viability as shown in Figure S7c. We therefore conclude that the mechanical process of injection does not significantly affect HeLa cell viability in the timeframe of the measurement.