L2 Slides - F24 Cell Biology PDF

Please log into PollEv using your NetID biomg1350fall24 https://pollev.com/biomg1350fall24 Cells under the microscope Reading: ECB6 1-39, 515-520 Learning Objectives: Understand size scales in cell biology Understand the uses of light microsc...

Please log into PollEv using your NetID biomg1350fall24 https://pollev.com/biomg1350fall24 Cells under the microscope Reading: ECB6 1-39, 515-520 Learning Objectives: Understand size scales in cell biology Understand the uses of light microscopy in cell biology Understand the uses of electron microscopy in cell biology Describe the major organelles of the cell  Size scales relevant to cell biology millimeter micrometer nanometer 1 mm = 10-3 m 1 mm = 10-6 m 1 nm = 10-9 m Figure 1-9 Essential Cell Biology  What is meant by Resolving Power (“Resolution”)? The ability to distinguish two close objects Consider two spots 0.1 µm (100 nm) apart Wavelength of illumination Light Microscope ~500 nm Electron Microscope ~0.003 nm 100x 1,000x 100,000x The maximum resolving power depends on the wavelength of the illumination. Visible light has a wavelength of 400-700nm and can resolve objects about 200nm apart. In a 200kV electron microscope the wavelength of the electrons is 3  Size scales relevant to cell biology 1000x 1000x Figure 1-9 Essential Cell Biology light microscope  Animal skin cell viewed by light microscopy Nucleus Bright field Panel 1-1 Essential Cell Biology Cells have thousands of different proteins. How to visualize a protein of interest? Human cells contain about 20,000 different proteins The abundance of each type of protein is different, some may be present in a few hundred copies, whereas others may be present in many million copies. How can we visualize a specific protein in a cell? Indirect immunofluorescence (IF) 1. Specificity Uses an antibody to your protein of interest, the ‘antigen’ UV/Blue Light Green Light 494 nm 521 nm 2. Sensitivity Fluorescence Solution of fluorescein A molecule is fluorescent if it absorbs light of one wavelength and then emits light of a different (longer) wavelength – very sensitive!  Indirect immunofluorescence (IF) First steps: ‘Fix’ cell (chemical “marker” is a fluorescent molecu cross-linking) and make sometimes called a “tag” cell permeable to antibodies using a detergent to dissolve the plasma membrane Wash away unbound antibodies Wash away unbound antibodies More details in active learning section next week Figure 9-18 Molecular Biology of the Cell Using IF to visualize actin in a culture cell Using IF to visualize actin (magenta) and tubulin (green)  Identifying Cancer Cells Using antibodies to a tumor cell marker, magenta fluorescence identifies cancer cells in a biopsy from a patient  Indirect immunofluorescence (IF) Can only be done by killing the cells! Wash away unbound antibodies! Wash away unbound Fix (crosslinking) antibodies! Permeabilize (detergent) Figure 9-18 Molecular Biology of the Cell  Green fluorescent protein (GFP) 1. Sensitivity GFP Fluorescence 2. Specificity Aequorea victoria GFP protein “fusion” Gene for protein of Gene for DNA interest GFP Green fluorescent protein (GFP) Protein Then express the fusion protein in cel  GFP-labeled proteins can be used to visualize organelles Can you guess, which organelle is shown here? Living cultured fibroblast imaged by fluorescence microscopy Transmission Electron Microscopes (TEM) For viewing thin samples in a vacuum Sample Fix Dehydrate And infiltrate with plastic section Stain the biological molecules with heavy metals that scatter electrons examine in vacuum  Liver cell viewed by TEM Figure 1-8A Essential Cell Biology Electron Microscopy “pros and cons” Light Microscopy and Electron Microscopy are complementary techniques EM “pro”: Much better resolution than light microscopy (more detail) EM “cons”: Much more effort to get 3D view of the cell (“tomography”) More difficult to label specific proteins Cannot be used on live cells (imaging is done in a vacuum)  What do we see when we look inside cells? Overview of cell organelles and cell structure Visualized organelles: ER (yellow) Golgi (magenta) peroxisomes (red) lipid droplets (blue) lysosomes (cyan) mitochondria (green) https://doi.org/10.1038/nature22369  Why do Eukaryotes contain internal membranes? Allows compartmentalization of function cells perform many competing reactions that need to be separated (ie. protein synthesis and protein degradation) Allows more membrane surface per cell volume many reactions are carried out on membranes larger cells have smaller ratio of cell surface to volume Where did the nucleus, endoplasmic reticulum, Golgi, endosomes, lysosomes, peroxisomes, etc. come from during evolution? We don’t really know! ECB6 shows a nice model (page 519) that might be true  The Plasma Membrane (PM) plasma membrane Function: Separates the cell from the environment Mediates interactions with the environment (signaling, nutrient uptake, endo- and exocytosis) Figure 1-24A Essential Cell Biology  Cytoplasm vs. Cytosol “Cytoplasm”: Everything in between the plasma membrane and the nucleus. “Cytosol”: Soluble portion of the cytoplasm outside of organelles Figure 1-24 Essential Cell Biology Cytosol is a very crowded environment Many chemical reactions including most of protein synthesis occur in the cytosol Model of the cytosol Function: Many chemical reactions including protein synthesis occur in the cytosol RNAs, proteins, and ribosomes are shown in different colors  The Nucleus Function: Contains the “genome” (most of cellular DNA) replication and transcription occur in the nucleus (darker regions are the “nucleolus” – this is where Section through a nucleus ribosomes are seen with an electron assembled) microscope Figure 1-15 Essential Cell Biology  Nuclear envelope and nuclear pores Nuclear pore nucleus cytoplasm Nuclear pore Section through a nucleus seen with an electron microscope Inner Outer membrane membrane of the nuclear ER membranes Figure 15-8B and 8C Essential Cell Biology  Endoplasmic reticulum (ER) Ribosomes Function: Primary site for Part of a cell showing Part of a cell showing the ER network by ER membranes with synthesis of lipids, fluorescence ribosomes by electron membrane proteins, microscopy microscopy and secreted Sections of ER with proteins ribosomes Figure 1-22 and 15-12 Essential Cell Biology bound are called ”rough pparatus (or “Golgi complex”, or simply “the G Function: Modification of secretory proteins Sorting station for vesicle trafficking Small area of a cell showing Figure 1-23 Essential Cell Biology the Golgi by electron pparatus (or “Golgi complex”, or simply “the G Golgi nucleus 10 mm Golgi by fluorescence Golgi by electron microscopy microscopy doi.org/10.1007/s00418-006-0166-5  Mitochondria Mitochondria have two membranes and their own genome Outer membrane Inner membrane Folded inner membran (“cristae”) A section through a mitochondrion imaged by Function: electron microscopy Major site for ATP production (“oxidative phosphorylation) Synthesis of iron-sulfur clusters Production of central metabolites (amino acids and Figure 1-18 nucleotides) Essential Cell Biology e structure of mitochondria – they are not bean (RFP) mitochondria nucleus Outer membrane Inner membrane Folded inner membrane (“cristae”) A section through a mitochondrion imaged by electron microscope Cristae contain the protein machinery for the generation of ATP  Mitochondria evolved from engulfed Theory bacteria of Endosymbiosis Mitochondria have their own genome and ribosomes. They are more closely related to bacterial genomes and ribosomes than to the Eukaryotic nuclear genome and ribosomes in the cytosol Figure 1-19 Essential Cell Biology  Chloroplasts “thylakoids” Cell isolated from aChloroplasts leaf perform photosynthesis Figure 1-20 Essential Cell Biology  Chloroplasts likely evolved from engulfed bacteria Chloroplasts have their own genome and ribosomes. They are more closely related to bacterial genomes and ribosomes than to the Eukaryotic nuclear genome and ribosomes in the cytosol Figure 1-21 Essential Cell Biology  ------- ~ 1 - 20 Table 15-2 Essential Cell Biology