The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in unders... more The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in understanding nuclear organization and function. Through the use of more natural tandem DNA arrays, Hu et al. (Hu, Y., I. Kireev, M. Plutz, N. Ashourian, and A.S. Belmont. 2009. J. Cell Biol. 185:87–100) gain new insights into chromatin organization and dynamics, and into the association of splicing factors with active genes.
Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known sys... more Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.
We developed an X-ray microscope using partially coherent object illumination instead of previous... more We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm (Rayleigh) and ∼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.
To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescen... more To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cel...
In vivo single molecule tracking has recently developed into a powerful technique for measuring a... more In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly differen...
In eukaryotic cells, post-translational histone modifications have an important role in gene regu... more In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our sin...
Dynamic molecular interactions are fundamental to all cellular processes. In vivo analyses of the... more Dynamic molecular interactions are fundamental to all cellular processes. In vivo analyses of these interactions are frequently done using fluorescence recovery after photobleaching (FRAP). Proper interpretation of FRAP data yields information about the binding interactions of fluorescently tagged molecules, including the number of binding states and the binding strength of each state. This binding information can be gleaned from appropriate models of the process underlying a FRAP recovery. Continued application and development of these approaches promise to provide crucial information for a quantitative description of the molecular networks that regulate cellular function.
During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressin... more During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting...
Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of fluorescently tagged mo... more Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of fluorescently tagged molecules within live cells. These molecular dynamics are governed by diffusion of the molecule and its in vivo binding interactions. As a result, quantitative estimates of the association and dissociation rates of binding can be extracted from the FRAP. This chapter describes a systematic procedure to acquire the FRAP data, and then fit it with appropriate mathematical models to estimate in vivo association and dissociation rates of binding. Also discussed are the applicability and limitations of the models, the utility of the estimated parameters, and the prospects for increased accuracy and confidence in the estimates.
FRET is a light microscopy method for detecting protein-protein interactions within intact cells.... more FRET is a light microscopy method for detecting protein-protein interactions within intact cells. The FRET protocol presented here is for CFP- and YFP-tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP-tagged protein (the FRET "acceptor") is photobleached at a cellular site of interest, and then the intensity of the CFP-tagged protein (the FRET "donor") at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP- and YFP-tagged proteins directly interact at the site of the photobleaching.
2012 9th IEEE International Symposium on Biomedical Imaging (ISBI), 2012
ABSTRACT In this simulation study, an engineered point-spread function (PSF) with reduced depth v... more ABSTRACT In this simulation study, an engineered point-spread function (PSF) with reduced depth variability (due to depth-induced aberrations) was applied to three-dimensional computational optical-sectioning microscopy (COSM) imaging to investigate its impact on image restoration. Intermediate synthetic images from PSF-engineered COSM in the presence of aberrations were computed and then processed using a depth-variant expectation maximization algorithm. The restored images were compared to images simulated for traditional COSM. Results show that PSF-engineered COSM achieves better image restoration than conventional COSM and has the potential to improve imaging in the presence of depth-induced aberrations.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2003
Protein interactions at the molecular level can be measured by fluorescence resonance energy tran... more Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency. A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by...
Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nucle... more Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nuclear protein binding to chromatin in live cells. FRAP can be applied qualitatively to assess if chromatin binding interactions are altered by various biological perturbations. It can also be applied semi-quantitatively to allow numerical comparisons between FRAP curves, and even fully quantitatively to yield estimates of in vivo diffusion constants and nuclear protein binding rates to chromatin. Here we describe how FRAP data should be collected and processed for these qualitative, semi-quantitative, and quantitative analyses.
The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in unders... more The binding of fluorescently tagged proteins to tandem DNA arrays has been instrumental in understanding nuclear organization and function. Through the use of more natural tandem DNA arrays, Hu et al. (Hu, Y., I. Kireev, M. Plutz, N. Ashourian, and A.S. Belmont. 2009. J. Cell Biol. 185:87–100) gain new insights into chromatin organization and dynamics, and into the association of splicing factors with active genes.
Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known sys... more Exchange of the glucocorticoid receptor (GR) at promoter target sites provides the only known system in which transcription factor cycling at a promoter is fast, occurring on a time scale of seconds. The mechanism and function of this rapid exchange are unknown. We provide evidence that proteasome activity is required for rapid GR exchange at a promoter. We also show that chaperones, specifically hsp90, stabilize the binding of GR to the promoter, complicating models in which the associated chaperone, p23, has been proposed to induce GR removal. Our results are the first to connect chaperone and proteasome functions in setting the residence time of a transcription factor at a target promoter. Moreover, our results reveal that longer GR residence times are consistently associated with greater transcriptional output, suggesting a new paradigm in which the rate of rapid exchange provides a means to tune transcriptional levels.
We developed an X-ray microscope using partially coherent object illumination instead of previous... more We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm (Rayleigh) and ∼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.
To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescen... more To investigate myosin II function in cell movement within a cell mass, we imaged green fluorescent protein-myosin heavy chain (GFP-MHC) cells moving within the tight mound of Dictyostelium discoideum. In the posterior cortex of cells undergoing rotational motion around the center of the mound, GFP-MHC cyclically formed a “C,” which converted to a spot as the cell retracted its rear. Consistent with an important role for myosin in rotation, cells failed to rotate when they lacked the myosin II heavy chain (MHC−) or when they contained predominantly monomeric myosin II (3xAsp). In cells lacking the myosin II regulatory light chain (RLC−), rotation was impaired and eventually ceased. These rotational defects reflect a mechanical problem in the 3xAsp and RLC− cells, because these mutants exhibited proper rotational guidance cues. MHC− cells exhibited disorganized and erratic rotational guidance cues, suggesting a requirement for the MHC in organizing these signals. However, the MHC− cel...
In vivo single molecule tracking has recently developed into a powerful technique for measuring a... more In vivo single molecule tracking has recently developed into a powerful technique for measuring and understanding the transient interactions of transcription factors (TF) with their chromatin response elements. However, this method still lacks a solid foundation for distinguishing between specific and non-specific interactions. To address this issue, we took advantage of the power of molecular genetics of yeast. Yeast TF Ace1p has only five specific sites in the genome and thus serves as a benchmark to distinguish specific from non-specific binding. Here, we show that the estimated residence time of the short-residence molecules is essentially the same for Hht1p, Ace1p and Hsf1p, equaling 0.12-0.32 s. These three DNA-binding proteins are very different in their structure, function and intracellular concentration. This suggests that (i) short-residence molecules are bound to DNA non-specifically, and (ii) that non-specific binding shares common characteristics between vastly differen...
In eukaryotic cells, post-translational histone modifications have an important role in gene regu... more In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our sin...
Dynamic molecular interactions are fundamental to all cellular processes. In vivo analyses of the... more Dynamic molecular interactions are fundamental to all cellular processes. In vivo analyses of these interactions are frequently done using fluorescence recovery after photobleaching (FRAP). Proper interpretation of FRAP data yields information about the binding interactions of fluorescently tagged molecules, including the number of binding states and the binding strength of each state. This binding information can be gleaned from appropriate models of the process underlying a FRAP recovery. Continued application and development of these approaches promise to provide crucial information for a quantitative description of the molecular networks that regulate cellular function.
During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressin... more During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting...
Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of fluorescently tagged mo... more Fluorescence recovery after photobleaching (FRAP) reveals the dynamics of fluorescently tagged molecules within live cells. These molecular dynamics are governed by diffusion of the molecule and its in vivo binding interactions. As a result, quantitative estimates of the association and dissociation rates of binding can be extracted from the FRAP. This chapter describes a systematic procedure to acquire the FRAP data, and then fit it with appropriate mathematical models to estimate in vivo association and dissociation rates of binding. Also discussed are the applicability and limitations of the models, the utility of the estimated parameters, and the prospects for increased accuracy and confidence in the estimates.
FRET is a light microscopy method for detecting protein-protein interactions within intact cells.... more FRET is a light microscopy method for detecting protein-protein interactions within intact cells. The FRET protocol presented here is for CFP- and YFP-tagged proteins examined with an argon laser on a scanning confocal microscope. FRET is assayed by one of the most straightforward approaches available, namely, acceptor photobleaching. In this procedure, the YFP-tagged protein (the FRET "acceptor") is photobleached at a cellular site of interest, and then the intensity of the CFP-tagged protein (the FRET "donor") at that same site is measured. In principle, FRET is detected when the CFP intensity increases after the photobleaching of YFP. This unit describes the appropriate steps to perform this measurement, as well as the necessary controls to ensure that an increase in CFP intensity, if detected, in fact reflects bona fide FRET. Successful application of the protocol will support the conclusion that the CFP- and YFP-tagged proteins directly interact at the site of the photobleaching.
2012 9th IEEE International Symposium on Biomedical Imaging (ISBI), 2012
ABSTRACT In this simulation study, an engineered point-spread function (PSF) with reduced depth v... more ABSTRACT In this simulation study, an engineered point-spread function (PSF) with reduced depth variability (due to depth-induced aberrations) was applied to three-dimensional computational optical-sectioning microscopy (COSM) imaging to investigate its impact on image restoration. Intermediate synthetic images from PSF-engineered COSM in the presence of aberrations were computed and then processed using a depth-variant expectation maximization algorithm. The restored images were compared to images simulated for traditional COSM. Results show that PSF-engineered COSM achieves better image restoration than conventional COSM and has the potential to improve imaging in the presence of depth-induced aberrations.
Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2003
Protein interactions at the molecular level can be measured by fluorescence resonance energy tran... more Protein interactions at the molecular level can be measured by fluorescence resonance energy transfer (FRET) using a pair of fluorescent proteins, such as CFP and YFP, in which the emission spectrum of CFP significantly overlaps the excitation spectrum of YFP. The resulting energy given off from the donor CFP protein can directly excite the acceptor YFP protein when the proteins are closely approximated. During FRET, there is quenching of the emission of the donor CFP protein that is directly related to the efficiency of energy transfer and inversely proportional to the sixth power of the distance between the donor and acceptor proteins. In this study we describe a new approach to visualize donor CFP quenching during CFP-->YFP FRET and demonstrate how this parameter can be used to calculate FRET efficiency. A novel flow cytometric method to detect protein-protein interactions in living cells was developed that utilized assessment of CFP donor quenching during CFP-->YFP FRET by...
Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nucle... more Fluorescence recovery after photobleaching (FRAP) has now become widely used to investigate nuclear protein binding to chromatin in live cells. FRAP can be applied qualitatively to assess if chromatin binding interactions are altered by various biological perturbations. It can also be applied semi-quantitatively to allow numerical comparisons between FRAP curves, and even fully quantitatively to yield estimates of in vivo diffusion constants and nuclear protein binding rates to chromatin. Here we describe how FRAP data should be collected and processed for these qualitative, semi-quantitative, and quantitative analyses.
Uploads
Papers by James McNally