Comparison of different methods for the isolation of Arabidopsis thaliana nuclear membranes

Year 2019, Volume: 9 Issue: 2, 681 - 691, 01.06.2019

Fatih Erci

https://doi.org/10.21597/jist.475545

Abstract

In animal and yeast system, subcellular fractionation has been widely used in studies of protein localization and organelle proteomics. Alternatively, it has not been an effective way of study in plant system because of some experimental limitations. The main aim of this study is to optimize subcellular and subnuclear fractionation of wild-type of Arabidopsis thaliana ecotype Colombia (Col-0) leaves by comparing three different methods to isolate nuclear membrane. In the study, we at first optimized nuclear washing steps to remove chloroplast contents from nuclear fractions. By optimizing speed of centrifugation and chemical component of the nuclear washing buffer, purified nuclear fractions was obtained. After measurement of protein amount for each fraction, purity of the fractions was analyzed by western blot assay with some specific cell compartment markers such as anti-Histone3 for nuclear fraction and anti-Rubisco for cytoplasmic fraction. Also, lactate dehydrogenase enzyme assay was used to confirm purity of the fractions. Then, subnuclear fractionation was done to isolate purified nuclear membrane. Three different methods were used to separate the nuclear membrane from whole purified nucleus. Nuclear compartment markers such as anti-Histone3 and anti-Fibrillarin was used in this step. In the study, Inner Nuclear Membrane protein (AtSUN2) was used as a nuclear membrane marker. All things considered, we conclude that the method with DNase digestion and high centrifugation speed (first method ) is a more effective way in separation of nuclear membrane fractions because the low centrifugal speed (second method) does not appear to be sufficient for separating the nuclear membrane, and the third method does not seem to be a very effective way as it requires a high centrifugal speed.

Keywords

Arabidopsis thaliana, Subcellular fractionation, Nuclear membranes

References

• Brandizzi F, Irons SL, Evans DE, 2004. The plant nuclear envelope: new prospects for a poorly understood structure. New phytologist, 163(2): 227-246.
• Boruc J, Zhou, Meier I, 2012. Dynamics of the plant nuclear envelope and nuclear pore. Plant Physiology, 158(1):78-86.
• Burke B, Roux KJ, 2009. Nuclei take a position: managing nuclear location. Developmental Cell, 17(5):587-597.
• Calikowski TT, Meulia T, Meier I, 2003. A proteomic study of the Arabidopsis nuclear matrix. Journal of cellular biochemistry, 90(2):361-78.
• Carmody SR, Wente SR, 2009. mRNA nuclear export at a glance. Journal of Cell Science, 122(12):1933-1937.
• Cheng YT, Germain H, Wiermer M, Bi D, Xu F, García A, Wirthmueller L, Després C, Parker JE, Zhang Y, Li X, 2009. Nuclear pore complex component MOS7/Nup88 is required for innate immunity and nuclear accumulation of defense regulators in Arabidopsis.The Plant Cell Online, 21(8):2503-2516.
• Ellis JA, 2006. Emery-Dreifuss muscular dystrophy at the nuclear envelope: 10 years on. Cellular and Molecular Life Sciences CMLS, 63(23):2702-2709.
• Graumann K, Runions J, Evans DE,2010. Characterization of SUN‐domain proteins at the higher plant nuclear envelope. The Plant Journal, 61(1):134-44.
• Haynes PA, Roberts TH, 2007. Subcellular shotgun proteomics in plants: looking beyond the usual suspects. Proteomics, 7(16):2963-2975.
• Hetzer MW, Walther TC, Mattaj IW, 2005. Pushing the envelope: structure, function, and dynamics of the nuclear periphery. Annual Review of Cell and Developmental Biology, 21:347-380.
• Kay RR, Fraser D, Johnston IR, 1972. A method for the rapid isolation of nuclear membranes from rat liver. Characterisation of the membrane preparation and its associated DNA polymerase. European Journal of Biochemistry / FEBS, 30(1):145-154.
• Kuznetsov AV, Gnaiger E, 2010. Laboratory Protocol Lactate Dehydrogenase Cytosolic Marker Enzyme. Mitochondrial Physiology Network, 8: 1-8.
• Mans B, Anantharaman V, Aravind L, Koonin EV, 2004. Comparative genomics, evolution and origins of the nuclear envelope and nuclear pore complex. Cell Cycle, 3(12):1625-1650.
• Matunis MJ, 2006. Isolation and fractionation of rat liver nuclear envelopes and nuclear pore complexes. Methods, 39(4):277-283.
• Meier I, Brkljacic J, 2009. Adding pieces to the puzzling plant nuclear envelope. Current Opinion in Plant Biology, 12(6):752-759.
• Meier I, 2007. Composition of the plant nuclear envelope: theme and variations.Journal of Experimental Botany, 58(1):27-34.
• Oda Y, Fukuda H, 2011. Dynamics of Arabidopsis SUN proteins during mitosis and their involvement in nuclear shaping.The Plant Journal, 66(4):629-641.
• Philipp EI, Franke WW, Keenan TW, Stadler J, Jarasch ED, 1976. Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue. The Journal of Cell Biology, 68(1):11-29.
• Piedras P, Rivas S, Droge S, Hillmer S, Jones JD, 2000. Functional, c-myc-tagged Cf-9 resistance gene products are plasma-membrane localized and glycosylated.The Plant Journal : For Cell and Molecular Biology, 21(6):529-536.
• Renner K, Amberger A, Konwalinka G, Kofler R and Gnaiger E. 2003. Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochimica Et Biophysica Acta (BBA)-Molecular Cell Research, 1642(1):115-123.
• Rodrigues MA, Gomes DA, Nathanson MH, Leite MF. 2009. Nuclear calcium signaling: a cell within a cell. Brazilian Journal of Medical and Biological Research, 42(1):17-20.
• Terry LJ, Shows EB, Wente SR, 2007. Crossing the nuclear envelope: hierarchical regulation of nucleocytoplasmic transport. Science Signaling, 318(5855):1412.
• Thuleau P, Brière C, and Mazars C, 2012. Recent advances in Plant cell Nuclear signaling. Molecular Plant, 5(5):968-970.
• Wheeler MA, Ellis JA, 2008. Molecular signatures of Emery-Dreifuss muscular dystrophy. Biochemical Society Transactions, 36(6):1354.
• Worman HJ, Bonne G, 2007. Laminopathies: a wide spectrum of human diseases. Experimental Cell Research, 313(10):2121-2133.