Abstract
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.
Highlights
High mobility group (HMG)-box containing proteins are ubiquitous non-histone nuclear protein found in eukaryotes
Fluorescence of enhanced green fluorescence protein (EGFP) fused to AtHMGB1, AtHMGB5 and AtHMGB14 were detected in nuclei and with diffused signal in the nucleolus (Fig 1)
EGFP fusion of HMGB proteins from Arabidopsis can imitate the localization of these proteins at interphase detected by immunolocalization study
Summary
High mobility group (HMG)-box containing proteins are ubiquitous non-histone nuclear protein found in eukaryotes. A number of plant HMGB proteins have been cloned from Arabidopsis [6,7,8], rice [9], maize [10,11], and cotton [12] These proteins were found involved in cell proliferation and differentiation [12], transcription [7,10], growth [13] and stress responses [12,13]. It is suggested that this characteristic can be a good indicator to distinguish HMGB proteins from the 3xHMG-box proteins which associates with condensed chromosome [1] These studies commonly adopted indirect immunofluorescence method which involved fixation with paraformaldehyde [14,15]. Localization of AtHMGB1 protein in metaphase cells was reinvestigated using enhance green fluorescence protein (EGFP) tagged proteins with or without paraformaldehdye fixation
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