Single-particle tracking of quantum dot-conjugated prion proteins inside yeast cells Toshikazu Tsuji a,1 , Shigeko Kawai-Noma a , Chan-Gi Pack b , Hideki Terajima a , Junichiro Yajima c , Takayuki Nishizaka c , Masataka Kinjo d , Hideki Taguchi a,⇑ a Department of Biomolecular Engineering, Graduate School of Biosciences and Biotechnology, Tokyo Institute of Technology, B56, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan b Cellular Informatics Laboratory, RIKEN Advanced Science Institute, Wako-shi, Saitama 351-0198, Japan c Department of Physics, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan d Laboratory of Molecular Cell Dynamics, Graduate School of Life Sciences, Hokkaido University, Sapporo 001-0021, Japan article info Article history: Received 8 January 2011 Available online 28 January 2011 Keywords: Quantum dots Single-particle tracking Yeast Yeast prion proteins abstract Yeast is a model eukaryote with a variety of biological resources. Here we developed a method to track a quantum dot (QD)-conjugated protein in the budding yeast Saccharomyces cerevisiae. We chemically con- jugated QDs with the yeast prion Sup35, incorporated them into yeast spheroplasts, and tracked the motions by conventional two-dimensional or three-dimensional tracking microscopy. The method paves the way toward the individual tracking of proteins of interest inside living yeast cells. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Understanding the dynamical behaviors of individual proteins of interest in living cells is one of the demands for cell biology in the post-genome era. Yeast cells are a quite attractive organism for monitoring individual protein motions, as budding and fission yeasts are model eukaryotes with a wide variety of biological re- sources, including vast amounts of global genetic and proteomic data, protein–protein interaction networks and so on [1]. Watching individual proteins inside cells usually requires the incorporation of proteins that are linked to a very bright fluorescent molecule, such as a quantum dot (QD) [2–4]. However, the incorporation of proteins from outside the yeast cells is considered to be difficult due to the very rigid yeast cell wall, although a unique ‘‘injection’’ method of proteins into fission yeast was recently reported, using a microfabrication system [5]. In the budding yeast Saccharomyces cerevisiae, the non-Mende- lian genetic elements such as [PSI + ] and [URE3] are typical prion- like protein-based genetic elements [6–10]. In [PSI + ] cells, the altered conformations of Sup35, which is the [PSI + ] determinant, are self-propagating aggregates and are transmitted to daughter cells [7–12]. Earlier single-cell approaches using fluorescence cor- relation spectroscopy (FCS) or fluorescence recovery after photo- bleaching (FRAP) revealed that the aggregated forms of Sup35 inside living cells are highly dynamic, in which continuous remod- eling of Sup35 oligomers are required to maintain and transmit the prion phenotype [13–19]. Since FCS and FRAP are ensemble meth- od for calculating the diffusion properties of fluorescent molecules [20,21], it cannot be used to unmask the individual behaviors of the molecules, which would provide unique insights into prion biology. To overcome the limitation of those ensemble techniques, here we developed a simple and versatile method to incorporate QD- conjugated proteins into living budding yeast using the yeast prion Sup35 as a model protein, and analyzed the individual motions of the Sup35-QD conjugates inside living yeast cells. 2. Material and methods 2.1. Protein expression and purification The DNA encoding SUP35NM was amplified by polymerase chain reaction (PCR) using pET-SUP35NMHC, which contains the N and M domains of the SUP35 gene [22], as a template. Sup35NHMC was purified in a similar manner as Sup35NMHC [22]. Further details are described in Supplementary Information. 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.01.083 Abbreviations: QD, quantum dot; FCS, fluorescence correlation spectroscopy; FRAP, fluorescence recovery after photobleaching; PEG, polyethylene glycol. ⇑ Corresponding author. Fax: +81 45 924 5785. E-mail address: taguchi@bio.titech.ac.jp (H. Taguchi). 1 Present address: Central Laboratories for Frontier Technology, Kirin Holdings Co., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. Biochemical and Biophysical Research Communications 405 (2011) 638–643 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc