( 343 nm DNA 483 nm) (5) ( ) 2. (Mitochondria) (mitochondrial genome) RNA (mrna) (matrix) (6) ( ) (signal sequence) (N-terminus) +H 3 N Met-Leu-Ser-Le

Size: px

Start display at page:

Download "( 343 nm DNA 483 nm) (5) ( ) 2. (Mitochondria) (mitochondrial genome) RNA (mrna) (matrix) (6) ( ) (signal sequence) (N-terminus) +H 3 N Met-Leu-Ser-Le"

往衲栾
7 years ago
Views:

1 Distributions of Cellular Proteins 1. (Nucleus) (transcriptional factors) (1) Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val- (Nuclear Location Signal, NLS) (2,3) NLS ( ) (nuclear import receptors) NLS (cargo proteins) (active transport) (nuclear pores) NLS 60,000 daltons 5,000 daltons (4) (cell transfections) (immunocytochemistry) ( ) DNA DAPI ( 359 nm DNA 461 nm) Hoechst

2 ( 343 nm DNA 483 nm) (5) ( ) 2. (Mitochondria) (mitochondrial genome) RNA (mrna) (matrix) (6) ( ) (signal sequence) (N-terminus) +H 3 N Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu- α-helix (protein translocators) (6) ATP 70 (heat shock protein, hsp 70) (signal peptidase) (7) c (cytochrome c oxidase) rhodamine 123 ( 504 nm 534 nm) Mito Tracker dyes (Molecular Probes) 70

3 (fixed) ( ( 1 mm) (5) 3. (rer) (post-translational modifications) (rough endoplasmic reticulum, rer) (Golgi apparatus) ( ) (N-terminus) +H 3 N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-G lu-gln-leu-thr-lys-cys-glu-val-phe-gln- (ER lumen) (8) (glycosylation) (9) (lysosome) (secretory vesicles) (plasma membrane) (epitope) 71

4 (secretory vesicles) (membranous organelles) DiO-C 6 (3) ( 573 daltons) ( 484 nm 501 nm) (5) DiO-C 6 (3) ER Tracker Blue-White DPX (Molecular Probes) 374 nm nm DiO-C 6 (3) ER ( ) 4. (Golgi apparatus) (lysosome) (secretory vesicles) (plasma membrane) ( ) (cis Golgi network) (trans Golgi network) ph (10) (histochemical stains) (acid phosphatase) (trans Golgi network) (ceramide) BODIPY-FL-ceramide (M.W. 576)(Molecular Probes) ( ) BODIPY-FL-ceramide 464 nm 532 (5) 5. (Lysosome) (acid hydrolases) (11) Neutral red acridine orange Neutral red 541 nm 640 nm (5) N-(3-[2,4-dinitrophenyl-amino]propyl)-N-(3-aminopropyl) methylamine (DAMP) LysoTracker LysoSensor probes (Molecular Probes) ( ) 72

5 T24 Hoechst MitoTracker Keratin 18 (Scale bar = 20 µm COS7 ER Tracker Blue-White DPX (Scale bar = 20 µm) 73

6 (GFP) COS7 BODIPY-FL-ceramide (A) GFP (B) (Scale bar = 20 µm) YFP-EB1 COS7 Hoechst LysoTracker YFP-EB1 (Scale bar = 20 µm) 74

7 COS7 Rhodamine Phalloidin (Scale bar = 20 µm) COS7 MAP1A (DsRed-HC, A) MAP1A (GFP-LC, B) AMCA (C) MAP1A (D) (Scale bar = 20 µm) 75

8 6. (Peroxisome) (C-terminus) -Ser-Lys-Leu-COO - ( ) (oxidative enzymes) catalase urate oxidase (hydrogen peroxide) (aldehyde) (phenol) (12) 7. (Cytoskeletons) (polyribosome) (13) (a) (Microfilaments) (actin) (F-actin) phallotoxins phalloidin phallotoxins ( Rhodamine 495 nm 520 nm) phalloidin (5) ( ) (b) (Intermediate filaments) 10 nm (14) (keratins) ( ) vimentin desmin (15) (c) (Microtubule) (tubulin) 23 nm (mitotic spindle) Paclitaxel (Taxol) (16) ( FITC) Paclitaxel 76

9 ( ) (Green Fluorescence Protein, GFP) (Jellyfish, Aequorea victoria) ( 488 nm 507 nm) ( ) (17, 18) (target gene) (fusion protein) ( 1999 (Discosoma striata) DsRed (BD Biosciences, Clontech) (19) ( 558 nm 583 nm) ( ) DsRed Clontech 2001 DsRed2 2 (Yellow Fluorescence Protein, YFP 513 nm 527 nm) ( ) (Cyanine Fluorescence Protein, CFP 433 nm 475 nm) DsRed 77

10 (confocal microscope) (20) ( 1. McKnight, S.L. and Yamamoto, K.R. (1992). Transcription Regulations. Cold Spring Harbor Laboratory Press, Plainview, NY, U.S.A. 2. Kalderon, D., Roberts, B.L., Richardson, W.D., and Smith, A.E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, Adam, S.A. (1999). Transport pathways of macromolecules between the nucleus and the cytoplasm. Curr. Opin. Cell Biol. 11, Wente, S.R. (2000). Gatekeepers of the nucleus. Science 288, Spector, D.L., Goldman, R.D., and Leinwand, L.A. (1998). Cells, A Laboratory Manual. Vol. 3, pp Cold Spring Harbor Laboratory Press, Plainview, NY, U.S.A. 6. Neupert, W. (1997). Protein import into mitochondria. Ann. Rev. Biochem. 66, Jensen, R.E. and Johnson, A.E. (1999). Protein translocation: is Hsp70 pulling my chain? Curr. Biol. 9, R779-R Mellman, I. and Warren, G. (2000). The road taken: past and future foundations of membrane traffic. Cell 100, Klumperman, J. (2000). Transport between ER and Golgi. Curr. Opin. Cell Biol. 12, Warren, G. and Malhotra, V. (1998). The organization of the Golgi apparatus. Curr. Opin. Cell Biol. 10, Andrews, N.W. (2000). Regulated secretion of conventional lysosomes. Trends. Cell Biol. 10, Tabak, H.F., Braakman, I., and Distel, B. (1999). Peroxisomes: simple in function but complex in maintenance. Trends Cell Biol. 9: Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. (2002). Molecular Biology of the Cell. 4th ed., pp Garland Science, New York, NY, U.S.A. 14. Fuchs, E. (1996). The cytoskeleton and disease: genetic disorders of intermediate filaments. Ann. Rev. Genet. 30, Spector, D.L., Goldman, R.D., and Leinwand, L.A. (1998). Cells, A Laboratory Manual. Vol. 3, 78

11 pp Cold Spring Harbor Laboratory Press, Plainview, NY, U.S.A. 16. Downing, K.H. (2000). Structural basis for the interaction of tubulin with proteins and drugs that affect microtubule dynamics. Ann. Rev. Cell Dev. Biol. 16, Heim, R., Cubitt, A.B., and Tsien, R.Y. (1995). Improved green fluorescence. Nature 373, Prasher, D.C. (1995). Using GFP to see the light. Trends Genet. 11, Baird, G.S., Zacharias, D.A., and Tsien, R.Y. (2000). Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA. 97, Paddock, S.W. (1999). Confocal Microscopy Methods and Protocols. Humana Press, Totowa, New Jersey, U.S.A. 79

12 80

i

i i ii 1 2 3 4 % N) 5 50.0 AMINO ACID #64 [modified by Administrator] IntAmp_1 nc 40.0 30.0 20.0 10.0 0.0-10.0 1 - ala - 10.483 2 - thr - 11.107 3 - gly - 11.872 4 - val - 13.458 5 - ser - 14.903 6 - pro