Paraformaldehyde Fixation May Lead to Misinterpretation of the Subcellular Localization of Plant High Mobility Group Box Proteins.
Arabidopsis High Mobility Group Box (HMBG) proteins were previously found associated with the interphase chromatin but not the metaphase chromosome. However, these studies are usually based on immunolocalization analysis involving paraformaldehyde fixation. Paraformaldehyde fixation has been widely adapted to preserved cell morphology before immunofluorescence staining. On one hand, the processed cells are no longer living. On the other hand, the processing may lead to misinterpretation of localization. HMGBs from Arabidopsis were fused with enhanced green fluorescence protein (EGFP) and transformed into tobacco BY-2 cells. Basically, the localization of these HMGB proteins detected with EGFP fluorescence in interphase agreed with previous publications. Upon 4% paraformaldehyde fixation, AtHMGB1 was found associated with interphase but not the metaphase chromosomes as previously reported. However, when EGFP fluorescence signal was directly observed under confocal microscope without fixation, association of AtHMGB1 with metaphase chromosomes can be detected. Paraformaldehyde fixation led to dissociation of EGFP tagged AtHMBG1 protein from metaphase chromosomes. This kind of pre-processing of live specimen may lead to dissociation of protein-protein or protein-nucleic acid interaction. Therefore, using of EGFP fusion proteins in live specimen is a better way to determine the correct localization and interaction of proteins.
Centre for Soybean Research of the Partner State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.
Man-Wah
MW
Li
Centre for Soybean Research of the Partner State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong; Department of Radiation Medicine, School of Public Health and Tropic Medicine, Southern Medical University, Guangzhou, Guangdong, People's Republic of China.
Liang
L
Zhou
Centre for Soybean Research of the Partner State Key Laboratory of Agrobiotechnology and School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong.
Hon-Ming
HM
Lam
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D032461:Chromosomes, Plant / Q000378:metabolism*; D005404:Fixatives / Q000494:pharmacology*; D005557:Formaldehyde / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D024243:HMGB1 Protein / Q000235:genetics / Q000378:metabolism*; D007399:Interphase; D008677:Metaphase; D011108:Polymers / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016707:Tissue Fixation / Q000379:methods; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D005404:Fixatives; D024243:HMGB1 Protein; C517312:HMGB1 protein, Arabidopsis; D011108:Polymers; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005557:Formaldehyde; C003043:paraform