Tài liệu Báo cáo khoa học: Analysis of the molecular dynamics of medaka nuage proteins by

Analysis of the molecular dynamics of medaka nuage

proteins by fluorescence correlation spectroscopy and

fluorescence recovery after photobleaching

Issei Nagao1,*, Yumiko Aoki2

, Minoru Tanaka2 and Masataka Kinjo1

1 Laboratory of Molecular Cell Dynamics, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan

2 Laboratory of Molecular Genetics for Reproduction, National Institute for Basic Biology, Okazaki, Japan

In most animals, primordial germ cells (PGCs) develop

distinctly from other cell lineages at a very early

embryonic stage, migrate towards the prospective

gonadal area, and then differentiate into gametes in

the gonads. Formation of the PGC requires germ

plasm, which contains electron-dense structures called

nuages that are believed to contain the determinants of

germ cells [1,2]. Although the nuage was reported half

Keywords

fluorescence correlation spectroscopy;

fluorescence recovery after photobleaching;

medaka; primordial germ cell; vasa

Correspondence

M. Kinjo, Laboratory of Molecular Cell

Dynamics, Faculty of Advanced Life

Science, Hokkaido University, Kita 21

Nishi 11, Kita-ku, Sapporo 001-0021, Japan

Fax: +81 1 706 9006

Tel: +81 1 706 9005

E-mail: [email protected]

*Present address

Biological Information Research Center,

National Institute of Advanced Industrial

Science and Technology (AIST) and Japan

Biological Informatics Consortium (JBIC),

Tokyo, Japan

Database

DNA data bank of Japan accession

numbers: olvas, AB063484; nanos3,

AB306931; tudor, AB306932

(Received 27 June 2007, revised 13

November 2007, accepted 21 November

2007)

doi:10.1111/j.1742-4658.2007.06204.x

The nuage is a unique organelle in animal germ cells that is known as an

electron-dense amorphous structure in the perinuclear region. Although the

nuage is essential for primordial germ cell (PGC) determination and devel￾opment, its roles and functions are poorly understood. Herein, we report

an analysis of the diffusion properties of the olvas gene product of the

medaka fish (Oryzias lapites) in PGCs prepared from embryos, using fluo￾rescence correlation spectroscopy and fluorescence recovery after photo￾bleaching. Olvas–green fluorescent protein (GFP) localized in granules

thought to be nuages, and exhibited a constraint movement with two-com￾ponent diffusion constants of 0.15 and 0.01 lm2

Æs

)1

. On the other hand,

cytosolic Olvas–GFP was also observed to have a diffusion movement of

7.0 lm2

Æs

)1

. Interestingly, Olvas–GFP could be expressed in HeLa cells,

and formed granules that were similar to nuages in medaka PGCs. Olvas–

GFP also exhibited a constraint movement in the granules and diffused in

the cytosol of HeLa cells, just as in the medaka embryo. The other two

gene products, Nanos and Tudor of the medaka, which are known as con￾stituents of the nuage, could also be expressed in HeLa cells and formed

granules that colocalized with Olvas–GFP. Nanos–GFP and Tudor–GFP

exhibited constraint movement in the granules and diffused in the cytosol

of HeLa cells. These results suggest that these granules in the HeLa cell are

not simple aggregations or rigid complexes, but dynamic structures consist￾ing of several proteins that shuttle back and forth between the cytosol and

the granules.

Abbreviations

CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; FAF, fluorescence autocorrelation function; FCS, fluorescence correlation

spectroscopy; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; LSM, laser scanning microscopy;

PGC, primordial germ cell; RFP, red fluorescent protein.

FEBS Journal 275 (2008) 341–349 ª 2007 The Authors Journal compilation ª 2007 FEBS 341