28. Visualizing Life - Fluorescent Proteins

MIT OpenCourseWare

12 May 202041:21

EducationalLearning

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TLDRThe script describes the discovery and impact of green fluorescent protein (GFP) from jellyfish. It explains how GFP revolutionized biological imaging by allowing scientists to tag proteins in living cells and observe them through fluorescence microscopy. The summary touches on key points like the structure of GFP, how it was engineered into different colors, and seminal experiments showing it could be expressed in organisms from bacteria to mice for imaging applications. Overall, it conveys how GFP enabled a major breakthrough in observing proteins dynamics in real time within living cells and organisms.

Takeaways

😀 Bioluminescence involves light emission from a biochemical reaction, often using the enzyme luciferase and substrate luciferin.
😃 The green fluorescent protein (GFP) was discovered in jellyfish and enables imaging of cells and proteins without fixing or staining.
😄 Martin Chalfie demonstrated GFP expression in organisms like C. elegans to enable live imaging of neurons and touch receptors.
🧐 The GFP fluorophore forms via cyclization of a Ser-Tyr-Gly sequence and oxidation to create a conjugated structure.
😯 Mutating the chromophore-forming Tyr residue alters GFP emission color; engineering cyan, blue, and yellow variants.
😲 Red fluorescent protein from coral extends the GFP chromophore to enable far-red emission.
🤓 GFP and RFP allow fluorescent labeling of organelles and proteins to study dynamics and localization in living cells.
😀 Live cell cycle imaging with different color FPs reveals timing of protein accumulation and degradation.
😊 GFP engineering created fruit FPs emitting across the spectrum, enabling multi-color cellular imaging.
😉 GFPs are versatile tools for tracking gene expression, protein localization, and cell processes in real time.

Q & A

What is bioluminescence?
-Bioluminescence is light emission that results from a biochemical reaction. A common example involves the enzyme luciferase reacting with the molecule luciferin in the presence of ATP to generate light.
What are DNA microarrays used for?
-DNA microarrays allow researchers to analyze expression of thousands of genes in parallel. They can be used to compare gene expression in healthy vs diseased cells to identify genes involved in disease.
What are the limitations of DNA microarrays?
-DNA microarrays measure mRNA levels, so they cannot detect problems that occur at the protein level, like defective translation or improper post-translational modifications.
How was GFP first discovered and characterized?
-GFP was first found in jellyfish by Shimamura. He crystallized it and solved its structure to understand how it fluoresces without added cofactors.
How did Martin Chalfie demonstrate the usefulness of GFP?
-Chalfie expressed GFP in model organisms like C. elegans and was able to visualize specific neurons and proteins, allowing real-time imaging in living cells and animals.
How was GFP engineered to create different fluorescent colors?
-By mutating key amino acids like Tyr in the GFP chromophore, variants emitting different wavelengths were created, like BFP and CFP.
Where did the red fluorescent protein dsRed come from?
-The red fluorescent protein dsRed was discovered in coral and was engineered to have an extended chromophore, resulting in longer wavelength emission.
How are fluorescent proteins enabling new imaging experiments?
-By fusing FPs to proteins of interest, researchers can label and visualize dynamics of specific proteins in living cells, like cell cycle regulation.
What makes fluorescent proteins superior to chemical dyes and antibodies for imaging?
-FPs allow labeling and live imaging without killing cells, unlike toxic chemical dyes. Also, FPs can be expressed inside cells, unlike antibodies.
What are some applications of fluorescent proteins in research?
-FPs are used as reporters of gene expression, to label specific proteins and organelles, and to visualize protein dynamics in living cells and model organisms.

Outlines

00:00

😊 Intro to Bioluminescence

The professor reminds students that bioluminescence is light emission from a biochemical reaction, often involving the enzyme luciferase and molecule luciferin. This occurs without an external light source. Bioluminescence assays are used in biological research.

05:03

🧪 Using DNA Arrays to Analyze Cells

The professor explains how DNA arrays allow rapid analysis of thousands of DNA sequences. Cells can be collected, mRNA isolated via the polyA tail, and cDNA synthesized with reverse transcriptase. Fluorophore labeling allows differential detection of cDNA from different cell populations when hybridized to the array.

10:05

❓ Limitations of DNA Arrays

The professor points out that while powerful, DNA arrays have limitations. A defective gene can still produce normal mRNA that binds the array. So protein-level defects would not be detected. Additional protein and functional experiments would be needed.

15:08

🤔 Why Antibodies Are Not Supravital

The professor explains that antibodies cannot be used to track live, viable cells over time. They disrupt cell function when binding surface proteins. And antibodies cannot permeate the plasma membrane to bind intracellular targets without first "fixing" (and killing) the cells.

20:09

🌟 Discovery and Properties of GFP

The professor discusses the discovery of green fluorescent protein (GFP) from jellyfish off the coast of Seattle. GFP needs no external cofactors to be fluorescent and revolutionized live imaging. The intrinsic fluorophore was characterized and modifications were made to improve GFP properties.

25:11

🔬 GFP Structure and Engineering New Colors

By examining the GFP structure and modifying the key tyrosine residue of the intrinsic fluorophore to tryptophan or phenylalanine via mutagenesis, researchers created new blue and cyan fluorescent protein variants, expanding the color palette available.

30:13

🐛 Using GFP to Label C. elegans Neurons

The professor shows how GFP allows fluorescent labeling of specific C. elegans neurons and touch receptors in live animals, enabling tracking of cells and proteins over time, unlike traditional antibody staining that requires sample fixing.

35:14

🎨 Creating a Spectrum of Fluorescent Proteins

Beyond GFP, new red, orange, and yellow fluorescent proteins were discovered or engineered by directed evolution and gene shuffling. This expanded toolkit allows differential labeling of multiple cellular components across the spectral range.

40:16

🧬 Observing Cell Dynamics with Fluorescent Proteins

The professor emphasizes how fluorescent proteins permit real-time imaging of chromosome dynamics, cell cycle regulation, protein stabilization, and more. This enables evaluating drug impacts on these processes to advance therapeutics.

Mindmap

Keywords

💡bioluminescence

Bioluminescence refers to light emission from a biochemical reaction within an organism. The video discusses different bioluminescent systems like the luciferase/luciferin reaction in mushrooms and the aequorin protein in jellyfish. Understanding bioluminescence helps explain the discovery of green fluorescent protein (GFP) in jellyfish and how it was adapted as a revolutionary imaging tool in biology.

💡GFP

GFP, or green fluorescent protein, was discovered in jellyfish off the coast of Seattle. It revolutionized imaging in biology because cells expressing GFP glow green under certain light without the need for any additional cofactors or substrates. The video details the structure of GFP, how its fluorophore forms, and early experiments tagging GFP to proteins in cells and organisms like C. elegans to observe them.

💡supravital dye

A supravital dye like DAPI can be used to stain and visualize live cells without killing them, unlike compounds like ethidium bromide. This makes supravital dyes critical for dynamic imaging experiments over time. The video contrasts DAPI against other fluorescent labels like antibodies in terms of their supravital capacities.

💡gene shuffling

Gene shuffling involves recombining DNA fragments from different parts of genes to engineer new protein variants. This was used to create fluorescent protein variants with different colors beyond GFP, like red, yellow, orange by shuffling genes from corals with the GFP gene.

💡protein engineering

The video discusses extensive protein engineering done on GFP to improve its properties. This included structure-guided changes like mutating two amino acid residues to prevent GFP dimerization and make it monomeric. Protein engineering expanded the utility of GFP and other fluorescent proteins enormously.

💡fixed cells

Unlike GFP and supravital dyes, antibody staining requires the use of fixed, dead cells. Fixing permeablizes membranes to allow antibody access inside the cells. Since fixed cells are static snapshots, imaging dynamic processes requires live staining methods like fluorescent proteins instead.

💡transcriptome

The video mentions that DNA microarrays are more useful for visualizing transcriptome changes between cell populations over the entire genome. The transcriptome representing all mRNA levels is a better indicator of cell state than just genomic differences alone.

💡promoter

By tagging GFP downstream of any promoter sequence in DNA, GFP acts as a reporter gene. If the promoter is active, GFP will be expressed and cells glow green. This provides an easy readout for testing promoter activity under different conditions.

💡fluorophore

The intrinsic fluorophore in GFP and other fluorescent proteins underlies their autofluorescent capabilities without external substrates. Understanding how this fluorophore structure forms and emits light informs protein engineering efforts to shift emitted wavelengths.

💡cell cycle

Fluorescent tagging of cell cycle regulatory proteins allows their levels to be dynamically monitored through different cell cycle phases. The video shows stunning imaging examples, discussing how certain arrests produce buildups of color that indicate the stalled phase.

Highlights

The study found a significant increase in life satisfaction for participants after the meditation program.

MRI scans showed changes in brain structure and connectivity after 8 weeks of meditation practice.

Meditation was linked to reduced stress, anxiety, and depression levels based on psychological assessments.

Participants reported improved emotional regulation and ability to cope with challenging situations.

The research provides evidence for meditation as an effective intervention for mental health issues.

No significant differences were found between visualization and mindfulness meditation techniques.

Experienced meditators showed greater changes in brain activity compared to beginners.

Limitations include a small sample size and lack of an active control group.

Further research with larger, randomized controlled trials is warranted.

Meditation may induce neuroplastic changes through repeated activation of relevant brain networks.

The underlying neurological mechanisms of meditation need to be explored further.

Individual differences in effects based on factors like personality traits should be examined.

The study provides a strong basis for future research on meditation and its clinical applications.

Widespread implementation of meditation-based interventions could benefit public mental health.

Meditation offers an accessible, cost-effective approach to improving well-being and treating mental health issues.

Transcripts

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