Optical Imaging:

polytene

Polytene Chromosomes
Formation of Polytene Chromosomes: The chromosomes are duplicated and stay connected and aligned. Patterns of denser “bands” alternate with lighter-staining “interbands” to form a consistently mappable genome.

Polytene chromosomes occur in the secretory glands of Drosophila (as well as in some other tissues and other dipteran flies). Multiple rounds of DNA replication result in about 1000 strands of DNA, which remain aligned and attached to each other. These chromosomes are large enough to view by light microscopy. Despite their unusual structure, polytenes function as interphase chromosomes and are transcriptionally active and responsive to hormonal and environmental signals (such as heat shock).

For more information on, and classical pictures of, polytene chromosomes, see also the Wikipedia article or the Sedat Lab description.

polytene immunofluorescnece
 
Spread Polytene Immunofluorescence
Immunofluorescence staining of spread polytene chromosomes: Two different proteins are pseudocolored red and green. Areas of colocalization between the two proteins appear yellow.

Indirect immunofluorescence staining of polytene chromosomes provides a rapid way of determining the in vivo genomic distribution of chromosomal proteins.

Polytenes are fixed and spread out on a slide, then probed with antibodies raised against specific proteins. This method allows us to visualize where proteins are in relationship to the underlying DNA sequence, and to each other.

The Drosophila genome has been sequenced, and the polytene maps have been correlated with the DNA sequence. This allows us to interpret staining patterns with precision, in some cases down to an individual gene.

Immunostaining for two or three factors at once allows comparison of factor distributions relative to one another. The colocalization of two factors may be indicative of a physical interaction in vivo.

Selected Papers:

  • Adelman K et al. (2005) “Efficient release from promoter-proximal stall sites requires transcript cleavage factor TFIIS.” Mol Cell. 17(1):103-12. (PubMed)
  • Schwartz BE et al. (2004) “Indirect immunofluorescent labeling of Drosophila polytene chromosomes: visualizing protein interactions with chromatin in vivo.” Methods Enzymol. 376:393-404. (PubMed)
  • Saunders A et al. (2003) “Tracking FACT and the RNA Polymerase II Elongation Complex Through Chromatin in Vivo.” Science 301: 1001-1140 (PubMed)
  • Boehm AK et al. (2003) “Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock.” Molecular and Cellular Biology23: 7628-7637. (PubMed)
Multiphoton Microscopy
Multiphoton microscopy 3-D reconstruction of a nucleus: HSF (in green) and DNA (in red) localization in a living polytene nucleus after heat shock.

Multiphoton Microscopy
Multiphoton microscopy 3-D reconstruction of a nucleus: HSF (in green) and DNA (in red) localization in a living polytene nucleus after heat shock.

In collaboration with Warren Zipfel’s Lab, we are using the optical sectioning power of multiphoton microscopy to examine protein localization, dynamics, and interactions at specific chromosomal loci in living polytene cells and in real time.

Proteins of interest are tagged with a fluorescent protein such as EGFP and transgenic lines that express this protein with temporal and spatial control can be created. Because of the ability of multiphoton microscopy to image deeply into a sample, the localization of the tagged protein can be watched in living cells in real time.

Additionally, the dynamics of the tagged protein can be studied using techniques such as FRAP (Fluorescence Recovery After Photobleaching) or FCS (Fluorescence Correlation Spectroscopy), and its interactions with another protein can be watched using FRET (Fluorescence Resonance Energy Transfer).

Selected Papers:

  • Zobeck KL, Buckley MS, Zipfel WR, and Lis JT. (2010) Recruitment Timing and Dynamics of Transcription Factors at the Hsp70 Loci in Living Cells. Molec Cell. 40: 965-975. (PubMed)
  • Yao J et al. (2007) “Intranuclear distribution and local dynamics of RNA polymerase II during transcription activation.” Mol Cell. 28(6):978-90. (PubMed)
  • Yao J et al. (2006) “Dynamics of heat shock factor association with native gene loci in living cells.”Nature. 442(7106):1050-3. (PubMed)