Heraud F E M S Lett

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First paper to put synchrotron IR light through a living algal cell.

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Heraud F E M S Lett

  1. 1. FEMS Microbiology Letters 249 (2005) 219–225 www.fems-microbiology.org Mapping of nutrient-induced biochemical changes in living algal cells using synchrotron infrared microspectroscopy Philip Heraud a,b,*, Bayden R. Wood a, Mark J. Tobin c, John Beardall a,b, Don McNaughton a a Centre for Biospectroscopy and School of Chemistry, Monash University, 3800 Vic., Australia b School of Biological Sciences, Monash University, 3800 Vic., Australia c Synchrotron Radiation Department, CCLRC Daresbury Laboratory, Warrington, Cheshire WA4 4AD, UK Received 28 February 2005; received in revised form 24 May 2005; accepted 2 June 2005 First published online 24 June 2005 Edited by G.M. Gadd Abstract High quality Fourier transform infrared (FTIR) spectra were acquired from living Micrasterias hardyi cells maintained in an IR transparent flow-through cell using a FTIR microscope coupled to a synchrotron light source. Spectral maps of living, nutrient- replete cells showed band intensities consistent with the known location of the nucleus and the chloroplasts. These were very similar to maps acquired from fixed, air-dried cells. Bands due to lipids were lowest in absorbance in the region of the nucleus and highest in the chloroplast region and this trend was reversed for the absorbance of bands attributed to protein. Spectra acquired in 10 lm steps across living phosphorus-starved (P-starved) cells, repeated approximately every 30 min, were consistent over time, and bands cor- related well with the known position of the nucleus and the observed chloroplasts, corroborating the observations with replete cells. Experiments in which missing nutrients were re-supplied to starved cells showed that cells could be maintained in a functional state in the flow-through cell for up to one day. Nitrogen-starved cells re-supplied with N showed an increase in lipid in all positions measured across the cell over a 23 h period of re-supply, with the largest increases occurring in positions where the chloroplasts were observed. Re-supply of phosphorus to P-starved cells produced no changes in bands attributable to lipid or protein. Due to their thin cell body ($12 lm) and large diameter ($300 lm) Micrasterias sp. make an ideal spectroscopic model to study nutrient kinetics in algal cells. Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Micrasterias; Synchrotron; IR spectroscopy; FT-IR; Microspectroscopy; Microalgae; Nutrient 1. Introduction essential for maintaining aquatic food chains and are important determinants in models describing global cli- Microalgae are the major primary producers in most mate change. It is not unusual for microalgae to become aquatic ecosystems, accounting for approximately 50% nutrient limited in the natural environment, particularly of total planetary primary productivity [1]. They are from nitrogen or phosphorus [2]. We have previously demonstrated that microalgal cells show dramatic reor- * ganization of macromolecular composition in responses Corresponding author. Tel./fax: +61 3 9905 4597. to changes in nutrient status, and this can be accurately E-mail address: phil.heraud@sci.monash.edu.au (P. Heraud). URL: http://web.chem.monash.edu.au/BioSpec/biospectroscopy. measured using Fourier transform infrared (FTIR) spec- html (P. Heraud). troscopy [3,4]. For example, starvation of nitrogen slows 0378-1097/$22.00 Ó 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2005.06.021
  2. 2. 220 P. Heraud et al. / FEMS Microbiology Letters 249 (2005) 219–225 protein synthesis and leads to the reduction of total pro- consisted of a 3 mm thick CaF2 lower window and a tein content of cells, and re-supply reverses these 2 mm thick ZnSe upper window separated by a 12 lm changes. Nutrient perturbations are ecologically impor- mylar spacer. JM medium was supplied continuously tant topics for study because they can alter the photo- by peristaltic pump, with the cuvette volume replaced synthetic efficiency of the organisms and alter their about 4 times per second. The field illumination from food quality to consumer organisms. the IR microscope was used to provide actinic light to In the above studies all measurements were per- the cells. Spectra were initially acquired from two P- formed on dehydrated cells and the observed changes starved cells that were re-supplied with nutrient-replete in response to nutrient perturbations were observed medium. Spectra were acquired: at 30, 60, 90, 105, after one day [3,4]. We hypothesized that more rapid 135, 165, 375,430, 555 and 615 min after re-supply of changes could be localized to different cellular compart- phosphorus to the P-starved cells. In a subsequent ments such as the nucleus or the chloroplasts. In this trial-replete medium was supplied to a nutrient-replete study, we employ the very intense flux of IR photons cell over 540 min, and the whole cell was mapped at provided by the synchrotron to explore the possibility 10 lm intervals. Spectral maps were also obtained from of locating, spatially and temporally, biochemical air-dried nutrient-replete cells deposited on a zinc sele- changes occurring within single living algal cells in re- nide substrate, using the 32· objective and a 10 lm2 sponse to nutrient perturbations. This study reports aperture, for comparison. Trials were then conducted the development of methodologies to analyze very large using N-starved cells. In these trials the cells were main- cells ($ 200 lm) from the genus Micrasterias as a model tained in the flow-through IR cell for almost one day. At organism. the end of this period the cells were removed and exam- ined by phase-contrast light microscopy. Measurements were taken 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 2. Materials and methods 330, 390, 596, 655 and 1380 min after re-supply of nitro- gen to the N-starved cell. 2.1. FTIR data collection 2.3. Spectral processing and data analysis FTIR absorption spectra were acquired using a Nic- olet Continuum microscope with SpectraTech 15· or All spectra were processed using OPUS NT 3.1 soft- 32· Reflchromat objectives attached to a Nexus FTIR ware (Bruker Optics, Ettlingen, Germany). Second spectrometer, which was connected to IR beamline derivative spectra, obtained using a Saviztky–Golay 11.1 at the Synchrotron Radiation Source (CCLRC smoothing function (13 points) and vector normalized Daresbury Laboratory, UK). Line mapping was to account for differences in sample thickness, were cal- performed across the center of cells, through the nuclear culated to minimize baseline differences and allow easy region, perpendicular to the isthmus between the semi- visual comparison. Integrated areas were calculated for cells, using the 15· objective and a 20 lm2 aperture with bands attributable to protein (amide II; 1565– a step length of 10 lm. At this magnification, a 20 lm2 1515 cmÀ1) and lipid (ester carbonyl; 1750–1720 cmÀ1). aperture allows the full available flux of the synchrotron to be focused onto the selected region of the cell. Previ- ous studies [5] have shown that biological samples un- 3. Results and discussion dergo negligible heating under similar conditions. Ten spectra were recorded in each line map at a spectral res- This study represents initial attempts to develop a olution of 8 cmÀ1 with 32 scans co-added. model system for in vivo analysis of single algal cells using synchrotron IR microspectroscopy. We have found 2.2. Preparation of cell samples M. hardyi cells to be ideal subjects for this work. They are large (up to 300 lm diameter) but thin (less than 12 lm) An axenic culture of Micrasterias hardyi (strain allowing them to be gently held in a cuvette with 12 lm CCAP649/15) was obtained from Sams Research Ser- spacers where they do not move when nutrient medium vices Ltd. (Dunstaffnage Marine Laboratory, Oban, is made to flow past them. The thin nature of the cell also Scotland). Cells were inoculated into either complete allows spectra to be recorded without spectral saturation. JM medium [6], JM medium lacking phosphorus, or Micrasterias cells appear to be easily maintained in a the same medium lacking nitrogen, and maintained functional state in the flow-through cuvette for periods for 4 days at 20 °C under continuous light of at least 1 day, provided they are provided with actinic (100 lm quanta mÀ2 sÀ1) provided by cool white fluo- light from the field illumination of the microscope and rescent tubes. Cells were placed in an IR-transparent the cuvette is continuously re-supplied with fresh medium flow-through cell, built in-house at SRS Daresbury, maintained at the growth temperature. This was verified based on a SpectraTech Demountable Flow Cell, which by cytoplasmic streaming being observed in cells
  3. 3. P. Heraud et al. / FEMS Microbiology Letters 249 (2005) 219–225 221 maintained in the IR flow-through cells for periods up to 24 h. Cytoplasmic streaming has been used previously as a test of viability in Micrasterias cells [7]. High quality spectra obtained using a 20 lm aperture on the IR microscope were very similar to spectra from air-dried cells (Fig. 1). The spectra were very detailed and contained absorption bands attributable to pro- teins, lipids, carbohydrates and phosphorylated mole- cules. The maximum of the amide I band at 1650 cmÀ1 corresponds to a high proportion of alpha helices com- pared to beta sheet secondary structure in the average protein content of the Micrasterias cells [8] (see Table 1). This corresponds with the known structure of ribu- lose bis-phosphate carboxylase (RUBISCO) and histone Fig. 2. Maps acquired at 10 lm intervals across nutrient-replete, air- dried and living M. hardyi cells. A 32· Cassegrain objective and a Fig. 1. Typical second derivative in vivo IR spectrum of M. hardyi 10 lm aperture on the IR microscope was employed to acquire spectra acquired using a 20 lm square aperture with a 15· objective, compared of the air-dried cell and a 15· objective and 20 lm aperture for the with a spectrum acquired using a 10 lm aperture in transmission from living cell. The images are contrasted using either the absorbance of the an air-dried M. hardyi cell with a 32· objective. The spectra shown are ester carbonyl band (1750–1720 cmÀ1) in the IR spectrum directly averages of 10 spectra acquired across the central axis of the cells at proportional to lipid concentration or the absorbance of the amide II 10 lm intervals. The major vibrational modes (minima) indicated are band (1565–1515 cmÀ1) in the IR spectrum directly proportional to explained in Table 1. protein concentration. Table 1 Band assignments for the IR spectrum of the in vivo M. hardyi cell Wavenumber Assignmenta Comments values (cmÀ1) $2970 mas(C–H) from methyl groups of lipids [16] $2920 mas(C–H) from methylene groups of lipids [16] $2850 ms(C–H) from methyl groups of lipids [16] $1740 m(C@O) of ester functional groups primarily from – lipids and fatty acids [18,19] $1650 Mainly m(C@O) associated with proteins[17,18] Referred to as the amide I band. May also contain contributions from C@C stretches of olefinic and aromatic compounds $1540 Mainly m(C–H) and d(N–H) associated with proteins [17,18] Referred to as the amide II band. May also contain contributions from C@N stretches $1455 das(CH3) and das(CH2) of proteins [20] $1370 ds(CH3) and ds(CH2) of proteins, and ds(C–O) of COOÀ groups [17,20] $1320 Mainly m(C–H) and d(N–H) of amides associated with proteins [21] Referred to as the amide III band $1240 mas(P@O) of the phosphodiester backbone of nucleic acid May also be due to the presence of phosphorylated (DNA and RNA) [17,22] proteins and polyphosphate storage products $1200–900 m(C–O–C) of polysaccharides [20,22] a mas, asymmetric stretch; ms, symmetric stretch; das, asymmetric deformation (bend); ds, symmetric deformation (bend).
  4. 4. 222 P. Heraud et al. / FEMS Microbiology Letters 249 (2005) 219–225 proteins, the major proteins found in the chloroplast in this flow-through cell configuration. Other commer- and nucleus, respectively, which have a high proportion cially available flow-through cells were trialled but all of alpha helices in their structure [9]. In this study the suffered from the same problem as the in-house built amide II band (1565–1515 cmÀ1) was used as an indica- unit used in this study. A new flow-through cell is cur- tor of protein concentration, because water absorbance rently being designed with a narrower housing to maxi- is lower in this region compared to the amide I [10]. Li- mize the working distance. A higher magnification pid concentration was determined using the integrated objective with a higher numerical aperture would im- area underneath the peak that is assigned to the ester prove the spatial resolution. carbonyl from lipids (1750–1720 cmÀ1). Despite the limitation of spatial resolution to 20 lm, We found that the signal-to-noise ratio deteriorated the very large size of the Micrasterias cell allowed us to significantly (data not shown) when apertures smaller resolve different compartments in the cell, the positions than 20 lm were used to analyze the living Micrasterias of which are well known from previous microscopic stud- cells. The 20 lm aperture is larger than the expected ies of M. hardyi cells [11]. The region of the cell close to wavelength dependent diffraction limited spatial resolu- the isthmus dividing the two semi-cells where the nucleus tion theoretically possible with synchrotron sources. The is known to reside [12] had a low concentration of lipid inability to obtain higher spatial resolution results partly but was high in protein. This is clear in the maps of dried from the lower power (15·) Cassegrain objective used in and living nutrient-replete cells (Fig. 2) and the lipid pro- this study, and from scattering and dispersive effects file across P-starved and N-starved cells (Figs. 3 and 4). within the organism being observed. Because of the lim- This corresponds to the known chemical composition ited working distance between the objective and the IR of the nucleus, which is very high in proteins (histones) flow-through cell, a 32· Cassegrain could not be used but very low in lipids. Conversely, regions where the Fig. 3. Temporal and spatial changes in lipid concentration and protein concentration in two phosphorus-starved M. hardyi cells over 615 min following resupply of phosphorus. Spectra were recorded through points along the central axis of the cells at 10 lm intervals using a 20 lm aperture on the IR microscope. The x-axis represents distance across the central axis of the cell; y-axis represents time after the re-addition of P; z-axis represents either the absorbance of the ester carbonyl band (1750–1720 cmÀ1) in the IR spectrum directly proportional to lipid concentration or the absorbance of the amide II band (1565–1515) in the IR spectrum directly proportional to protein concentration. The image of the M. hardyi cell was acquired from the video camera attached to the IR microscope at the start of the experiment. The positions at which spectra were acquired are indicated by the grid across the central axis of the cell.
  5. 5. P. Heraud et al. / FEMS Microbiology Letters 249 (2005) 219–225 223 Fig. 4. Temporal and spatial changes in lipid concentration and protein concentration in two nitrogen-starved M. hardyi cells over 1380 min following resupply of nitrogen. Spectra were recorded through points along the central axis of the cells at 10 lm intervals using a 20 lm aperture on the IR microscope. The x-axis represents distance across the central axis of the cell; y-axis represents time after the re-addition of N; z-axis represents either the absorbance of the ester carbonyl band (1750–1720 cmÀ1) in the IR spectrum directly proportional to lipid concentration or the absorbance of the amide II band (1565–1515 cmÀ1) in the IR spectrum directly proportional to protein concentration. There is a marked increase in ester carbonyl absorbance at the peripheries of both cells, measuring 20% and 38% at a position 30 lm from the edge of cell 1 and 2, respectively. The image of the M. hardyi cell was acquired from the video camera attached to the IR microscope at the start of the experiment. The positions at which spectra were acquired are indicated by the grid across the central axis of the cell. chloroplasts were observed correspond to regions of high species of microalgal cells that had been starved of P lipid concentration (Figs. 2–4), possibly due to the dense [4], which declined over a period of days after P was packing of membranes (thylakoids) known to exist in the re-supplied to the cells. No change in lipid concentration chloroplasts. The regions where chloroplasts were ob- was observed in any of the P-starved Micrasterias cells served in nutrient-replete and P-starved cells were also tested that were re-supplied with P (Table 2). Evidently, protein rich (Figs. 2 and 3), an observation which can these changes, if they occur, are not registered within the be explained by the high protein concentration known 10 h after re-supply of P during which cells were ob- to occur in the chloroplast. However, the protein concen- served. We intend to extend the time period over which tration of cytoplasmic regions in cells was not as high as observations are made in an attempt to observe changes that registered in the nuclear region. The profile of chem- induced by the re-introduction of P. ical concentration of lipid and protein across the cells Nitrogen starvation caused a decrease in protein was very consistent from measurement to measurement, absorbances (Fig. 4) compared with nutrient-replete corroborating the validity of the technique. The differ- cells (Fig. 2 and Table 2). When nitrogen was re- ence in concentration of lipid and protein between the supplied to starved cells an increase in lipid ester carbonyl nuclear region and the chloroplast region is shown to absorbance was observed over a 23 h period in both cells be significantly different, as shown in Table 2 for a num- (Fig. 4), consistent with the re-synthesis of this macro- ber of nutrient-replete and P-limited cells; regions which molecular class. The increases appear to be localized coincide with the chloroplasts are rich in lipid and the within the chloroplast region and may represent synthe- nuclear region is poor in lipid, whereas the nuclear region sis of lipids in the plastids. There was little change in has a higher concentration of protein compared with the amide II absorbance over the period of re-supply. chloroplast region. We have previously reported increases in protein P-starved cells had higher lipid content than nutrient- concentration after the re-supply of N to starved mic- replete cells (cf. Figs. 2 and 3 and Table 2). We have pre- roalgal cells [3], but not an increase in lipids. For viously observed a build up of lipid in a number of example, diatoms have been observed to build up lipid
  6. 6. 224 P. Heraud et al. / FEMS Microbiology Letters 249 (2005) 219–225 Table 2 cells and chloroplasts [15]. By comparing biochemical Showing the mean relative absorbances of the ester carbonyl band and physiological changes, we aim to obtain a richer from lipids and the amide II band from protein measured: at a central position in two P-starved cells (60 lm from the right-hand edge of the understanding of the dynamic interactions occurring be- cells along the line shown in Fig. 3), and two N-starved cells (50 lm tween cells and the environment. from the right-hand edge of the cells along the line shown in Fig. 4); compared with mean relative absorbances of the bands measured at a peripheral position in the same cells (40 lm from the right-hand edge of the P-starved cells and 30 lm from the right-hand edge of the N- References starved cells) Peripheral Central P [1] Shelly, K., Heraud, P. and Beardall, J. (2002) Nitrogen limitation position position in Dunaliella tertiolecta (Chlorophyceae) leads to increased susceptibility to damage by UV-B radiation but also increased Mean SD Mean SD repair capacity. J. Phycol. 38, 1–8. Lipid ester carbonyl absorbance [2] Harris, G.P. (1986) Phytoplankton Ecology, Structure, Function Replete cell 0.34a 0.01 0.23a 0.01 <0.005 and Fluctuation. Chapman & Hall, London. P-starved cell1 0.52b 0.01 0.37b 0.02 <0.02 [3] Giordano, M., Kansiz, M., Heraud, P., Beardall, J., Wood, B. P-starved cell 2 0.94c 0.01 0.55c 0.04 <0.001 and McNaughton, D. (2001) Fourier transform infrared spec- N-starved cell 1 0.59b 0.01 0.35b 0.01 <0.001 troscopy as a novel tool to investigate changes in intracellular N-starved cell 2 0.29a 0.01 0.23a 0.01 0.06 macromolecular pools in the marine microalga Chaetoceros muellerii (Bacillariophyceae). J. Phycol. 37, 271–279. Protein amide II absorbance [4] Beardall, J., Berman, T., Heraud, P., Kadiri, M., Light, B., Replete cell 0.22a 0.01 0.29a 0.01 0.08 Patterson, G., Roberts, S., Sahan, E., Schulzberger, B., Urlin- P-starved cell 1 0.27a 0.01 0.39b 0.01 <0.01 ger, U. and Wood, B. (2001) A comparison of methods for P-starved cell 2 0.37b 0.01 0.51c 0.01 <0.002 detection of phosphate limitation in microalgae. Aquat. Sci. 63, N-starved cell 1 0.12c 0.01 0.11d 0.01 n.s. 107–121. N-starved cell 2 0.06d 0.01 0.08e 0.01 n.s. [5] Martin, M., Tsvetkova, N.M., Crowe, J.H. and McKinney, W.R. Measurements represent the means of the repeated measurements (2001) Negligible sample heating from synchrotron infrared beam. (n = 22) at these positions in the cells in the nutrient re-supply exper- Appl. Spectrosc. 55, 111–113. iments. These results are compared with in vivo measurements from [6] Thomkins, J., DeVille, M.M., Day, J.G. and Turner, M.F., Eds., the nutrient-replete cell shown in Fig. 2. The results presented are the (1995). Culture Collection of Algae and Protozoa, Catalogue of mean from six spectra acquired from the central region compared with Strains. Natural Environmental Research Council, Ambleside, the mean of six spectra recorded from the region where the chloro- 204pp. plasts were observed. SD is the standard deviation on the means and P [7] Meindl, U. and Lutz, C. (1996) Effects of UV irradiation on cell ¨ is the probability of the difference between the means in the peripheral development and ultrastructure of the green alga Micrasterias. J. and central position occurring by chance alone (ANOVA). For a given Photochem. Photobiol. B 36, 285–292. column, values with different superscripts are significantly different by [8] Byler, D.M. and Susi, H. (1986) Examination of the secondary ANOVA (a = 0.05). structure of proteins by deconvolved FTIR spectra. Biopolymers 25, 469–487. [9] Laskowski, R.A., Hutchinson, E.G., Michie, A.D., Wallace, under N-starvation, which is reversed when N is re- A.C., Jones, M.L. and Thornton, J.M. (1997) PDBsum: a Web- supplied [3]. The decline in lipids observed under based database of summaries and analyses of all PDB structures. N-starvation in M. hardyi must represent a decline in Trends Biochem. Sci. 22, 488–490. the synthesis of lipids induced by starvation. We intend [10] Flach, C.R., Brauner, J.W., Taylor, J.W., Baldwin, R.C. and to study and attempt to elucidate this response more Mendelsohn, R. (1994) External reflection FTIR of peptide monolayer films in situ at the air/water interface: experimental fully. design, spectra–structure correlations, and effects of hydrogen– The Micrasterias cells appear to be robust enough to deuterium exchange. Biophys. J. 67, 402–410. be kept in good condition for at least a day, allowing for [11] Meindl, U. (1993) Micrasterias cells as a model system for a broad range of cellular responses to environmental research on morphogenesis. Microbiol. Rev. 57, 415–433. stressors, which are known to take place over periods [12] Meindl, U., Zhang, D. and Hepler, P.K. (1994) Actin microfilaments are associated with the migrating nucleus and of hours, to be monitored. These include responses to the cell cortex in the green alga Micrasterias. J. Cell Sci. 107, ultraviolet radiation exposure [13] and changes in tem- 1929–1934. perature [14] amongst many others. We intend to refine [13] Heraud, P. and Beardall, J. (2000) Changes in chlorophyll these experiments by using a new flow-through IR- fluorescence during exposure of Dunaliella tertiolecta to UV transparent cell to improve spatial resolution and study radiation indicate a dynamic interaction between damage and repair processes. Photosynth. Res. 63, 123–134. possible responses to the re-supply of phosphorus and [14] Schreiber, U., Bilger, W. and Neubauer, C. (1994) Chloro- other nutrients over the longer time interval of one phyll fluorescence as a non-intrusive indicator for rapid day that we now know is possible to achieve. Further- assessment of in vivo photosynthesis In: Ecophysiology of more, we would like to combine the IR measurements Photosynthesis (Caldwell, M.M., Ed.), pp. 49–70. Springer, of biochemical change with newly developed apparatus Berlin, Germany. [15] Goh, C.H., Schreiber, U. and Hedrich, R. (1999) New approach that allow physiological changes to be measured at the of monitoring changes in chlorophyll a fluorescence of single single cell level such as the microscopy-PAM, which guard cells and protoplasts in response to physiological stimuli. can measure photosynthetic electron transport in single Plant Cell Environ. 22, 1057–1070.
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