BioSci D103: Cell Biology

rofessors:

Cell Biology (Bio D103)

Dr. Shin Lin
shinlin@uci.edu
Adam Tuttle Elizabeth Gray atuttle@uci.edu graye@uci.edu

Dr. Steven Gross

sgross@uci.edu

eaching Assistants: Michelle Mattson mkmattso@uci.edu

Cos-7 cells Nikon MicroscopyU

Class Information
Class Website: https://eee.uci.edu/11f/05520 Syllabus Lecture schedule and lecture notes (.pdf) Animations and outside links FAQ

Contact: Email to sgross@uci.edu or shinlin@uci.edu or TAs Please use proper e-mail etiquette (see class website) Include Bio D103 in subject line Include your name and SID Use UCI e-mail accounts (no Hotmail etc.)
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Cell Biology lecture 1 Gross - all rights reserved.

Class Information
Grades Exam and course grading will be curved EXAM 1 (Midterm): 40 % EXAM 2 (Final, NOT comprehensive): 40 % Online quizzes/hw: 9 % 1/week Helps students get regular feedback to know where they are In-class iClicker questions: 10 % 2-3 questions every lecture, allowed to miss 2 lectures of clicker input. Absolutely no make-ups. Zero tolerance for missing clicker input (didnt work, missed day, etc). We do not have enough support to do anything else.

iClicker STARTS SEPT. 30.

Class evaluation: 1% Class etiquette: be polite, dont come in more than a minute or two late, dont leave early, turn off cell phones.
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Cell Biology lecture 1 Gross - all rights reserved.

Class Information
Exams NO MAKE-UP EXAMS FOR ANY REASON. GET INCOMPLETE GRADE AND RETAKE EXAM IN A SUBSEQUENT QUARTER Web-board
Very importantask questions and have them answered Supervision by TAs Potential extra credit if active in answering questions correctlyhelpful in borderline grade cases Problem-based learning sessions ~ 1/week, given by the TAs Opportunity for more in-depth discussion and consideration 5

Cell Biology lecture 1 Gross - all rights reserved.

Class Information (Dr. Lin)

Powerpoint Slides Slides presented in lecture will be posted on website approximately once a week. In the meantime, look at slides posted on website for D103 Fall 2009 (90+% similar). Some slides will not be discussed in class but assigned for after class reading (same as text reading as below) Text Book Readings Read to understand materials presented in Powerpoint slides Readings assigned in lectures, covering materials not on slides or on slides not covered in lecture due to time limitation. For deeper understanding of CELL BIOLOGY to improve performance in subsequent courses and tests (e.g., MCAT) Recommendation Letters Because of the large size of this class, Dr. Lin will regrettably not be able to write letters for any one (such impersonal letters dont count anyway)

Cell Biology lecture 1 Gross - all rights reserved.

Lecture 1

Introduction to Cell Biology.

Research Tools & Approaches.
Recommended Reading : MBOC (5th edition) 579-615

Molecular

Biology

Cell Biology

Organismal Biology

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Why Study Cell Biology?

Understanding how a single cell work can lead to the understanding of how a complex, multi-cellular organism works Essential for understanding and treatment of human diseases
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The key to every biological problem must be sought in the cell, for every organism is, or at sometime has been, a cell. Edward Beecher Wilson, Cell Biologist, 1925
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Organism = total of all organ systems working together System =several different organs working together to perform a major bodily function Organ = cooperative union of multiple tissue types working together to perform a single function Tissue = a group of similar cells working together to perform a specific function

Cell = smallest living unit of the body

Molecular = molecules are made of atoms covalently bound together Atoms = smallest (?) stable units of matter
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Approaches to Biology
Biochemistry: molecular structure, function, and chemistry of purified cell constituents Genetics: roles of healthy genes and consequences of damaged genes Developmental Biology: how cells change as they specialize Cell Biology: structure and function of cells and their components

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What Does Cell Biology Study?

Structure and function of the cell
organelles and molecular components interactions with other cells and with environment cell cycle, division, and death

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How is Cell Biology Studied?

Microscopic Molecular

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Size Matters!

Electron microscope

Light microscope
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What Limits What We Can See by Light Microscopy?

Visible spectrum

Rule of thumb for the resolving power: 1/2 wavelength resolution limit of light microscopy: 200 nm

How can we see smaller structures???

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What Can Be Seen by Electron Microscopy?

Structures as small as 0.2 nm can be resolved by electron microscopy.

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Information Obtained by Microscopy

Microscopy provides detailed information on how something looks (size, number) where something localizes Microscopy provides some information on the function of a given structure or protein the concentration of a protein/factor the mechanism of how a cellular process works --> biochemical/molecular biological approaches for more detailed information
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Types of Microscopy
Light microscopy Fluorescence microscopy

Transmission electron microscopy Scanning electron microscopy

Atomic force microscopy

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Fixing and Semi-Specific Staining of Cells for Bright Field Microscopy

Cells Stained with Hemtoxylin for Nucleic Acids and Eosin for Cytoplasmic Protein
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What Can Be Seen in Living Cells by Light Microscopy?

Bright Field

Phase Contrast

Nomarski Differential Interference Contrast

Darkfield

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 Fluorescent dyes: for example, DAPI is a DNA probe Fluorescent dyes can be coupled to antibodies to serve as highly selective and specific staining reagents Multiple dyes of different color can be used to detect different components in the cell Common fluorescent dyes include
Fluorescein: green Rhodamine: red Alexa dyes Cy3, cy5

Specific Molecules Can Be Located in Cells by Fluorescence Microscopy

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Principle of Fluorescence Microscopy

Grey: Fluorophore Spectra Blue:Exciter Filter Red: Emitter Filter Green: Dichroic Filter

Fluorescent molecules absorbs light at one wavelength and emit it at another, longer wavelength

Fluorescence microscopy is most often used to detect specific proteins or other molecules in the cells
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Anatomy of a Fluorescence Microscope

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Immunofluorescence

The fluorescent signal can be amplified by labeling the secondary antibody with fluorescent dyecalled indirect immunofluorescence
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A cell in mitosis Spindle microtubules are revealed with a green fluorescent antibody, centromere with a red fluorescent antibody, and the DNA with the blue fluorescent dye DAPI
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Human Lung Carcinoma Cells These cancerous epithelial cells have been utilized in a wide array of research, especially in scientific studies of viral infections associated with asthma, asbestosrelated tissue damage, emphysema, and other respiratory problems. Blue: cytokeratin, pink: mitochondria, green: DNA
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Principle of Confocal Microscopy

CONVENTIONAL
Confocal microscopy offers several advantages over conventional optical microscopy: (1) shallow depth of field, (2) elimination of out-of-focus glare, and (3) the ability to collect serial optical sections from thick specimens.
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CONFOCAL

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Confocal Microscopy
Light is focused at a single spot (rather than the whole specimen) Out of focus fluorescence is excluded Data from each point in the plane of focus are collected. 3-dimensional image can be reconstructed by scanning through the depth of the specimen

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Cos-7 cells Nikon MicroscopyU

Transformed (Simian Virus 40) African Green Monkey Kidney Fibroblast Cells Red: microtubules, green: actin, blue: DNA
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Bovine Pulmonary Artery Endothelial Cells Endothelial cells are cells of the inner lining of blood vessels. These cells are widely used in studies relating to hypertension, atherosclerosis, and coronary heart disease. Red: mitochondria, green: actin, blue: DNA
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Fluorescent Proteins Can Be Used to Tag Individual Proteins in Living Cells and Organisms
Green fluorescent protein (GFP)
isolated from a jellyfish variants with different color generated can be introduced into live cells as a reporter molecule transgenic organism can be made with the GFP sequence placed under the transcriptional control of the promoter of a gene of interest
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Digital Video Microscopy of Living Cells

Mitosis VIDEO (Fluorescent protein labeling of microtubules and histones showing mitotic spindle fibers pulling chromosomes apart during metaphase of mitosis)

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Different Types of Electron Microscopy (I)

Thin-section of a cell
Hepatitis B virus particles

Frozen-fractured muscle cell

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DNA
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Molecular Microscopy

Different Types of Electron Microscopy (II)

(A)Scanning electron microscopy showing the structure of stereocillia of inner ear

(B) Shown by DIC light microscope (C) Shown by thin-section transmission electron microscope
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Atomic Force Microscopy

AFM can give information: (a) sub-nanometer scale, (b) biomechanical properties, (c) reaction to mechanical perturbation.
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Model Systems in Cell Biology Research

Simple system to study a biological question for extrapolation of results to higher organisms Avoid human subject concerns Availability of genetics 3 types of systems: prokaryotic cell single cell eukaryote multicellular organisms
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All Cells are Prokaryotic or Eukaryotic

Eukaryotic features: Compartmentalization Cytoskeleton

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Compartments (Organelles) In Eukaryotic Cell

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Cell Culture Systems

Cells can be grown on specially treated glass/plastic plates or in suspension in defined/semi-defined media for experimental manipulations.
1959 Eagles Minimum Essential Medium: amino acids, salts, glucose, vitamins. Modified Essential Medium Supplementation with fetal calf serum (growth factors) 1970s Satos mix of defined factors.

Development of specialized surfaces and structures by/for tissue engineering (e.g., biocompatible, 3D scaffolding, bioreactive, bioerodable).

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Cell Culture Systems

Primary Culture
Mouse Keratinocytes from skin
Electrical Signals Direct Cell Migration in Wound Healing and Activate Selected Signaling Pathways
Zhao et al., Nature 442, 457-460(2006) MOVIE 1

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 Primary Culture

Cell Culture Systems

Human cells from biopsy of tumors

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 Permanent (Immortal) Cell Lines

Cell Culture Systems

HeLa Cells
Cervical cancer cells from Henrietta Lacks (1940s)

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Developmental Cell Biology

All Cells Arise From Pre-existing Cells

Human egg (1 cell) + sperm (1 cell) 10 trillion cells!

The key to every biological problem must be sought in the cell, for every organism is, or at sometime has been, a cell. Edward Beecher Wilson, Cell Biologist, 1925 43

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Developmental Biology & Stem Cell Biology First Cell Divisions Result in Pluripotent Stem Cells

followed by subsequent specialization (differentiation)

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What Determines a Scientists Choice of a Single Cell to Multi-Cell Model Organism?

Yeast 5,800 genes Fruit fly 13,000 genes Nematode (worm) 19,000 genes Mouse 30,000 genes

Haploid mutants and deletions can easily be generated, great for genetics and for screens
Easy and cheap to grow, Fast doubling time

Diploid Genetics, screens for components

Diploid Genetics, screens for components

Diploid Genetics are possible, gene deletion mutants and transgenic animals can be generated
Not so easy anymore!! Development, immunology, mammalian genetics and 45 cell 45 biology

Relatively easy Relatively easy and and cheap to grow cheap to grow Precise timing of development from single cell to adult with 959 cells, cell biology

Basic cell biological Development from and genetic questions single cell to multicellular organism, cell D103 Fall 2009 Lecture 1 Lin All rights reserved biology

Basic Techniques for Studying Gene Expression

Protein Level RNA Level

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Immunological Staining Techniques

Western Blotting
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Immunofluorescence Microscopy
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Hybridization Techniques (I) Northern and Southern Blots

Northern Blot: Detection of RNA with labeled DNA probe

Southern Blot: Detection of DNA with labeled DNA probe (Originally named after Edwin Southern)
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Hybridization Techniques (II) Fluorescence In Situ Hybridization

FISH Technique can be used for localization of both DNA and RNA
In situ-Hybridization of wild type Drosophila embryos at different developmental stages for the RNA from a gene called hunchback. 49 D103 Fall 2011 Lecture 1 Lin All rights reserved

Modern Experimental Techniques

Genomics

Reveals differences in structure and expression of entire genomes

Techniques: DNA Microarrays Monitor global patterns of gene expression Detect all the mRNAs present in a cell (identify which genes are active) What can Microarrays tell us? Liver cells transcribe a different set of genes than do white blood cells or skin cells Changes in gene expression during Disease progression In response to drugs During development

Proteomics
Monitors the presence and interactions of numerous proteins simultaneously Monitors the state and change in cellular metabolites

Metabolomics

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