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Following Molecules/Cells through TIME to Understand
Processing and Processes
Experimental strategies for investigating:
1. Kinetics of synthesis or degradation of a molecule
2. Precursor/product relationships
3. Molecular mechanisms (e.g., DNA replication, signal transduction)
4. Movements of a molecule over time (protein secretion, cell cycle, differentiation)
5. Which cells give rise to particular structures during development?
1. Need
2. Need
Experimental Conditions
1. Need a means of differentially marking a population of molecules or cells.
2. Need
Experimental Conditions
1. Need a means of differentially marking a population of molecules or cells.
2. Need a method for following them through time. Must distinguish labeled from unlabeled at various time points.
Experimental Conditions
1. Molecules
2. Cells
For marking:
1. Moleculesa. Radioactivity (e.g., 3H, 35S, 32P)
b. Density
c. Fluorescent or enzyme tags (with chromogenic substrates)
2. Cells
For marking:
1. Moleculesa. Radioactivity (e.g., 3H, 35S, 32P)
b. Density
c. Fluorescent or enzyme tags (with chromogenic substrates)
2. Cellsa. Fluorescent or enzyme tags (with
chromogenic substrates) – expressed or microinjected
b. Morphology (e.g., chick versus quail)
For marking:
1. The label must not affect the process of interest
2. There must be minimal redistribution of the label over the course of the experiment (except as produced by the process of interest)
3. (For some experiments) Labeling must be rapid (rapid cellular uptake and incorporation).
Marking constraints:
1. Radioactivity
2. Density
3. Fluorescence and enzyme labeling
For detecting/tracking:
1. Radioactivitya. Cell/molecule fractionation and counting
b. Microscopy (autoradiography)
2. Density
3. Fluorescence and enzyme labeling
For detecting/tracking:
1. Radioactivitya. Cell/molecule fractionation and counting
b. Microscopy (autoradiography)
2. Densitya. Density-gradient centrifugation
b. Mass spectrometry
3. Fluorescence and enzyme labeling
For detecting/tracking:
E. Milner et al., 2006
E. Milner et al., 2006
Section of a coral tentacle
Epiderm
Nematocysts
Gastroderm
Dinoflagellates
Mesentery
1. Radioactivitya. Cell/molecule fractionation and counting
b. Microscopy (autoradiography)
2. Densitya. Density-gradient centrifugation
b. Mass spectrometry
3. Fluorescence and enzyme labelinga. Microscopy
b. Cell/molecular fractionation and observation (of fractions, gels, chromatograms)
For detecting/tracking:
Density-gradient centrifugationComes in two flavors!
Density-gradient centrifugationComes in two flavors!
1. Velocity centrifugation
2. Equilibrium (isopycnic) centrifugation
Density-gradient centrifugationComes in two flavors!
1. Velocity centrifugationMaterials: Sucrose, Ficoll (low osmolarity), etc.
Procedure: Premix gradient, run for fixed time
Separation: Depends on mass, shape, partial-specific volume (density), which determine “S value”
Density-gradient centrifugationComes in two flavors!
1. Velocity centrifugationMaterials: Sucrose, Ficoll (low osmolarity), etc.
Procedure: Premix gradient, run for fixed time
Separation: Depends on mass, shape, partial-specific volume (density), which determine “S value”
2. Equilibrium (isopycnic) centrifugationProcedure: Run to equilibrium
Separation: Depends only on density (neutral buoyancy)
a. “Step gradients” (pre-form gradient of sucrose, etc.)
b. “Continuous gradients” [gradient forms itself by sedimentation vs. diffusion; CsCl (nucleic acids), Percoll (cells and organelles)]
Meselson & Stahl (1958)One of the most famous experiments ever – why?
Meselson & Stahl (1958)One of the most famous experiments ever – why?
• They solved an important problem (did DNA replicate the way Watson’s & Crick’s model predicted?).
Meselson & Stahl (1958)One of the most famous experiments ever – why?
• They solved an important problem (did DNA replicate the way Watson’s & Crick’s model predicted?).
• They pioneered use of stable-isotope labeling AND isopycnic density-gradient centrifugation in CsCl (with Vinograd).
Meselson & Stahl (1958)One of the most famous experiments ever – why?
• They solved an important problem (did DNA replicate the way Watson’s & Crick’s model predicted?).
• They pioneered use of stable-isotope labeling AND isopycnic density-gradient centrifugation in CsCl (with Vinograd).
• Methods were elegant, they attended punctiliously to detail (craftsmanship!), the results were very clear, and the presentation was lucid (more craftsmanship!).
conservative distributive
semi-conservative
4000 kb X 650 kDa/kb ≈ 2.6 X 109
How would you answer this question today?
Look directly at the DNA molecule?Resolution is an issue. Maybe atomic force microscopy?
What about BrdU labeling? Resolution wouldn’t be good enough to distinguish strands.
Here rapid labeling IS an issue.Often, cells are first grown in a metabolite-deficient medium to deplete their stores of that metabolite.
Pulse/Chase Experiments
Here rapid labeling IS an issue.Often, cells are first grown in a metabolite-deficient medium to deplete their stores of that metabolite.
Labeled metabolite is added, or a tagging procedure is applied, for a discrete interval (the “pulse”).
Pulse/Chase Experiments
Here rapid labeling IS an issue.Often, cells are first grown in a metabolite-deficient medium to deplete their stores of that metabolite.
Labeled metabolite is added, or a tagging procedure is applied, for a discrete interval (the “pulse”).
Cells are then washed and/or an excess of unlabeled metabolite is added (the “chase”).
Pulse/Chase Experiments
Here rapid labeling IS an issue.Often, cells are first grown in a metabolite-deficient medium to deplete their stores of that metabolite.
Labeled metabolite is added, or a tagging procedure is applied, for a discrete interval (the “pulse”).
Cells are then washed and/or an excess of unlabeled metabolite is added (the “chase”).
Cells are sampled at intervals to track the metabolite (or tagged molecule) and molecules and/or organelles into which it is incorporated.
Pulse/Chase Experiments
Curr. Biol. 18:1203-1208 (2008)