We present a single-cell motility assay, which allows the quantification of bacterial swimming in... more We present a single-cell motility assay, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of ∼100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we
We present two dynamical models for the appearance of adaptive mutations, which permit a quantita... more We present two dynamical models for the appearance of adaptive mutations, which permit a quantitative comparison with experimental observations. The models quantify two alternative pictures: The hyper-mutating sub-population picture, andthe population-wide mutation picture. We #nd that both models are equally successful in reproducing the results of experiments in which a single mutation is required. They also enable us to estimate the values of key biological parameters, such as successful mutation rates. We then analyze the multiple mutations experiment of Rosche and Foster (Proc. Natl. Acad.
Base-pairing interactions between nucleic acids mediate target recognition in many biological pro... more Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.
Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage ... more Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in β-galactosidase levels in mutT lacZamber cultures, without invoking participat...
Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar... more Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its...
Physical review. E, Statistical, nonlinear, and soft matter physics, 2009
We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability... more We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability theory, showing that the repressor circuit comprises four redundant components whose failure mode is prophage induction. Our model reflects the specific biochemical mechanisms involved in regulation, including long-range cooperative binding, and its detailed predictions for prophage induction in E. coli under ultraviolet radiation are in good agreement with experimental data.
The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a... more The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination 1,2 . Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant) 3,4 . We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells 5 . Here, we describe the full procedure for performing the infection experiments described in our earlier work 5 . This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild-type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection 6 . The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.
Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Alth... more Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell's flagella cooperatively determine whether the cell 'runs' or 'tumbles' remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell's run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an 'effective number' of flagella-smaller than the actual number-enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella.
Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-inter... more Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation.
Studying the 3D sub-cellular structure of living cells is essential to our understanding of biolo... more Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a noninvasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.
Studying the 3D sub-cellular structure of living cells is essential to our understanding of biolo... more Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. In contemporary cell microscopy the most common technique to address this problem is through the use of fluorescence microscopy techniques [1]. Although several fluorescence techniques, including several superresolution methods, have been developed to address the problem, they require specialized probes complicated experimental setups and heavy trade-offs between resolution and field of view in space and time [2]. On the other hand ...
Robustness is a property built into biological systems to ensure stereotypical outcomes despite f... more Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/a-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-a (Sry-a) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-a are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-a collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-a and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.
We present a single-cell motility assay, which allows the quantification of bacterial swimming in... more We present a single-cell motility assay, which allows the quantification of bacterial swimming in a well-controlled environment, for durations of up to an hour and with a temporal resolution greater than the flagellar rotation rates of ∼100 Hz. The assay is based on an instrument combining optical tweezers, light and fluorescence microscopy, and a microfluidic chamber. Using this device we
We present two dynamical models for the appearance of adaptive mutations, which permit a quantita... more We present two dynamical models for the appearance of adaptive mutations, which permit a quantitative comparison with experimental observations. The models quantify two alternative pictures: The hyper-mutating sub-population picture, andthe population-wide mutation picture. We #nd that both models are equally successful in reproducing the results of experiments in which a single mutation is required. They also enable us to estimate the values of key biological parameters, such as successful mutation rates. We then analyze the multiple mutations experiment of Rosche and Foster (Proc. Natl. Acad.
Base-pairing interactions between nucleic acids mediate target recognition in many biological pro... more Base-pairing interactions between nucleic acids mediate target recognition in many biological processes. We developed a super-resolution imaging and modeling platform that enabled the in vivo determination of base pairing-mediated target recognition kinetics. We examined a stress-induced bacterial small RNA, SgrS, which induces the degradation of target messenger RNAs (mRNAs). SgrS binds to a primary target mRNA in a reversible and dynamic fashion, and formation of SgrS-mRNA complexes is rate-limiting, dictating the overall regulation efficiency in vivo. Examination of a secondary target indicated that differences in the target search kinetics contribute to setting the regulation priority among different target mRNAs. This super-resolution imaging and analysis approach provides a conceptual framework that can be generalized to other small RNA systems and other target search processes.
Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage ... more Living in an oxygen-rich environment is dangerous for a cell. Reactive oxygen species can damage DNA, RNA, protein and lipids. The MutT protein in Escherichia coli removes 8-oxo-deoxyguanosine triphosphate (8-oxo-dGTP) and 8-oxo-guanosine triphosphate (8-oxo-GTP) from the nucleotide pools precluding incorporation into DNA and RNA. While 8-oxo-dGTP incorporation into DNA is mutagenic, it is not clear if 8-oxo-GTP incorporation into RNA can have phenotypic consequences for the cell. We use a bistable epigenetic switch sensitive to transcription errors in the Escherichia coli lacI transcript to monitor transient RNA errors. We do not observe any increase in epigenetic switching in mutT cells. We revisit the original observation of partial phenotypic suppression of a lacZamber allele in a mutT background that was attributed to RNA errors. We find that Lac+ revertants can completely account for the increase in β-galactosidase levels in mutT lacZamber cultures, without invoking participat...
Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar... more Many bacteria are motile only when nutrients are scarce. In contrast, Salmonella enterica serovar Typhimurium is motile only when nutrients are plentiful, suggesting that this bacterium uses motility for purposes other than foraging, most likely for host colonization. In this study, we investigated how nutrients affect motility in S. enterica and found that they tune the fraction of motile cells. In particular, we observed coexisting populations of motile and nonmotile cells, with the distribution being determined by the concentration of nutrients in the growth medium. Interestingly, S. enterica responds not to a single nutrient but apparently to a complex mixture of them. Using a combination of experimentation and mathematical modeling, we investigated the mechanism governing this behavior and found that it results from two antagonizing regulatory proteins, FliZ and YdiV. We also found that a positive feedback loop involving the alternate sigma factor FliA is required, although its...
Physical review. E, Statistical, nonlinear, and soft matter physics, 2009
We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability... more We analyze a system-level model for lytic repression of lambda phage in E. coli using reliability theory, showing that the repressor circuit comprises four redundant components whose failure mode is prophage induction. Our model reflects the specific biochemical mechanisms involved in regulation, including long-range cooperative binding, and its detailed predictions for prophage induction in E. coli under ultraviolet radiation are in good agreement with experimental data.
The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a... more The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination 1,2 . Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant) 3,4 . We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells 5 . Here, we describe the full procedure for performing the infection experiments described in our earlier work 5 . This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild-type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35°C to trigger viral DNA injection 6 . The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.
Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Alth... more Bacterial chemotaxis is a paradigm for how environmental signals modulate cellular behavior. Although the network underlying this process has been studied extensively, we do not yet have an end-to-end understanding of chemotaxis. Specifically, how the rotational states of a cell's flagella cooperatively determine whether the cell 'runs' or 'tumbles' remains poorly characterized. Here, we measure the swimming behavior of individual E. coli cells while simultaneously detecting the rotational states of each flagellum. We find that a simple mathematical expression relates the cell's run/tumble bias to the number and average rotational state of its flagella. However, due to inter-flagellar correlations, an 'effective number' of flagella-smaller than the actual number-enters into this relation. Data from a chemotaxis mutant and stochastic modeling suggest that fluctuations of the regulator CheY-P are the source of flagellar correlations. A consequence of inter-flagellar correlations is that run/tumble behavior is only weakly dependent on number of flagella.
Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-inter... more Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation.
Studying the 3D sub-cellular structure of living cells is essential to our understanding of biolo... more Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. However, tomographic imaging of live cells is challenging mainly because they are transparent, i.e., weakly scattering structures. Therefore, this type of imaging has been implemented largely using fluorescence techniques. While confocal fluorescence imaging is a common approach to achieve sectioning, it requires fluorescence probes that are often harmful to the living specimen. On the other hand, by using the intrinsic contrast of the structures it is possible to study living cells in a noninvasive manner. One method that provides high-resolution quantitative information about nanoscale structures is a broadband interferometric technique known as Spatial Light Interference Microscopy (SLIM). In addition to rendering quantitative phase information, when combined with a high numerical aperture objective, SLIM also provides excellent depth sectioning capabilities. However, like in all linear optical systems, SLIM's resolution is limited by diffraction. Here we present a novel 3D field deconvolution algorithm that exploits the sparsity of phase images and renders images with resolution beyond the diffraction limit. We employ this label-free method, called deconvolution Spatial Light Interference Tomography (dSLIT), to visualize coiled sub-cellular structures in E. coli cells which are most likely the cytoskeletal MreB protein and the division site regulating MinCDE proteins. Previously these structures have only been observed using specialized strains and plasmids and fluorescence techniques. Our results indicate that dSLIT can be employed to study such structures in a practical and non-invasive manner.
Studying the 3D sub-cellular structure of living cells is essential to our understanding of biolo... more Studying the 3D sub-cellular structure of living cells is essential to our understanding of biological function. In contemporary cell microscopy the most common technique to address this problem is through the use of fluorescence microscopy techniques [1]. Although several fluorescence techniques, including several superresolution methods, have been developed to address the problem, they require specialized probes complicated experimental setups and heavy trade-offs between resolution and field of view in space and time [2]. On the other hand ...
Robustness is a property built into biological systems to ensure stereotypical outcomes despite f... more Robustness is a property built into biological systems to ensure stereotypical outcomes despite fluctuating inputs from gene dosage, biochemical noise, and the environment. During development, robustness safeguards embryos against structural and functional defects. Yet, our understanding of how robustness is achieved in embryos is limited. While much attention has been paid to the role of gene and signaling networks in promoting robust cell fate determination, little has been done to rigorously assay how mechanical processes like morphogenesis are designed to buffer against variable conditions. Here we show that the cell shape changes that drive morphogenesis can be made robust by mechanisms targeting the actin cytoskeleton. We identified two novel members of the Vinculin/a-Catenin Superfamily that work together to promote robustness during Drosophila cellularization, the dramatic tissue-building event that generates the primary epithelium of the embryo. We find that zygotically-expressed Serendipity-a (Sry-a) and maternally-loaded Spitting Image (Spt) share a redundant, actin-regulating activity during cellularization. Spt alone is sufficient for cellularization at an optimal temperature, but both Spt plus Sry-a are required at high temperature and when actin assembly is compromised by genetic perturbation. Our results offer a clear example of how the maternal and zygotic genomes interact to promote the robustness of early developmental events. Specifically, the Spt and Sry-a collaboration is informative when it comes to genes that show both a maternal and zygotic requirement during a given morphogenetic process. For the cellularization of Drosophilids, Sry-a and its expression profile may represent a genetic adaptive trait with the sole purpose of making this extreme event more reliable. Since all morphogenesis depends on cytoskeletal remodeling, both in embryos and adults, we suggest that robustness-promoting mechanisms aimed at actin could be effective at all life stages.
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Papers by Ido Golding