Biophysics I (PC2267)

The roles of Physics in Biological systems

Lecturer: Assist/prof. Yan Jie Physics DepartmentOffice: S7-01-10Phone: 65162620Course information and lecture notes:http://www.physics.nus.edu.sg/~Biophysics/BiophyiscsSyllabusa.htmlPassword: biopc2267

Physics background check statistics

•We have 40 students registered, 30 of them responded to the survey.

•Twenty six know some calculus, one doesn’t know, and three chose “the others”.

•Twenty two know some mechanics, three doesn’t, five chose “the others”.

•Twenty six know some thermal physics, three doesn’t know, and one chose “the others”.

•Eight know some statistical physics, twenty doesn’t know, and two chose “the others”.

•Twenty are from Physics Department, ten from other departments.

Calculus is required by the course. At least you should know the concept of Integral,

Contents of course

• Outline of the biology and physics relevant to the course

• Introduction to the thermal physics and statistical physics need to understand the course

• Introduction to random walk, friction, and diffusion

• Applications of the physics learnt to understanding various biological systems

Course plan and evaluationI will use 3 hours for lecture, and one hour for tutorial each week.

There will be several (~4) homework assignments.

Your grades will be determined by:

Mid-term exam: 20%Final exam: 30%Experiments attendance + reports: 20% Class attendance 10%Homework: 20%

Please contact our lab officer Mr. Teo Hoon Hwee (phythh) and Mr. Low Yee Teck (phylyt) for lab arrangment.

The discussion and viva in lab sessions will be conducted by Dr. Du Ning (phydn) and Dr. Zhang Keqin (phyzkq).

Cell and the central dogma of molecular biology

Cell structure, cell molecules, gene expression, and cell cycle

Copied for teaching purpose only from:Lodish, Berk/Matsudaira/Kaiser/Krieger/Scott, Molecular Cell Biology

Please see more details in the supplementary page at the end of the lecture note.

Simple facts of the cells•Cell is the basic unit of the life•Cell needs energy input to maintain its function and activity•Cell synthesizes macromolecules (like proteins) to satisfy its needs to grow and function•Cell can divide itself, during the process the genetic material is also duplicated•Cell can maintain a voltage difference across the cell membrane. This is done by maintain a difference of the concentration of ions between the inside and outside of the cell•Many cells can move•Cell can sense the environment and make corresponding responses•Inside the cell, there exists a complex regulation mechanism to keep the cell contents to be healthy and normal•In certain cases, as part of the regulation system, cell can suicide

Molecules of the cell•Small molecules: simple sugars, amino acids, vitamins, and so on

•ATP is the best known small molecule , which stores the energy.•Cell breaks down food molecules and stores the energy released into ATP molecules.•Plant cell and a few other organism can harvest energy in sunlightto form ATP in photosynthesis.

•Some small molecules act as Signals within and between cells , e.g., hormone and neuro-transmitters•Certain small molecules (monomers) can be linked to form polymers (macromolecules)

• Macromolecules: polymers made of linked monomers•Polysaccharides are made of linked sugars.•Proteins are made of several hundred of 20 different linked Amino Acids, contributing 20% cell weight.•Nucleic Acids (DNAs and RNAs ) are made of linked nucleotides,carrying the genetic information.

•Ions: sodium, chloride, calcium, and So on

base pairs: A-T & C-G

Proteins are polymers of amino acids linked together in a particular order specified by a gene's DNA sequence.

Proteins perform a wide variety of functions in the cell; these include serving as enzymes, structural components, or signaling molecules.

DNA is a polymer of nucleotides. Nucleotides are further composed of a phosphate group, a sugar, and a nitrogenous base.

DNA Contain genetic information.

Genes are DNA sequences coded for making proteins

Steps involved:

•DNA Replication

•Gene transcription into MRNA

•mRNA migration

•Gene translation into proteins

http://www.accessexcellence.org/RC/VL/GG/central.html

Copied for teaching purpose only Recorded by my previous labmate, now a Professor at OSU. Dr. Michael Poirier.

Lots of physics behind the picture!

Movie: cell division Live!

Cell MitosisMitosis: a process of cell division which results in the production of two daughter cells.

Mitosis can be divided into four principals stages:

Prophase: The chromatin, diffuse in interphase, condenses into chromosomes. Each chromosome has duplicated and now consists of two sister chromatids. At the end of prophase, the nuclear envelope breaks down into vesicles.

Metaphase: The chromosomes align at the equitorial plate and are held in place by microtubules attached to the mitotic spindle and to part of the centromere.

Anaphase: The centromeres divide. Sister chromatids separate and move toward the corresponding poles.

Telophase: Daughter chromosomes arrive at the poles and the microtubules disappear. The condensed chromatin expands and the nuclear envelope reappears.

Cytokinesis: The cytoplasm divides, the cell membrane pinches inward ultimately producing two daughter cells .

Break

What physics can contribute?

•Physics plays its roles in almost every cell process described in previous slides.

•Physicists has been contributing to the understanding of life from very beginning of the discovery of DNA.

•In modern biology, physicists are still contributing to the understanding of the fundamental questions in cell biology.

•Biophysicists are doing research complimentary to what pure Biologists are doing.

•Biophysicists are also developing novel techniques that can be used by Biologists.

Below I will impress you with examples of real state-of-art research of cell biology conducted by Biophysicists.

Experimental instruments used by Biophysicists

Introduction to the powerful single-molecule imaging and

manipulation techniques

Single-DNA imaging using AFM

• Direct structural and conformational information of chromatin

• Detect the nucleosome distribution on chromatin• Detect biochemical dependence of the higher

order structure of chromatin

Assembly using purified proteins

Bennink group

AFM imaging technique

DNA bending by IHF proteinYan lab

Single-DNA fluorescence imaging

Streptavidin coated quantum dots

Biotinylated anti-nucleosome antibody

Fluorescence imaging

• Direct information about compaction of DNA and nucleosome formation dynamics

• Same idea applies to the study of the activities of motor proteins processing on chromatin template

DNA stained by YOYO-1Yan lab

A. Crut et el

Means of single DNA manipulations and applications

T. Strick et al.,Physics Today,page 46October, 2001

a. Glass fiber b. Atomic Force Microscopec. Optical tweezerd. Magnetic tweezera,b,c use deflection of the force sensors to measure forces, d uses the fluctuation of bead to measure forces

Virtues of MT:a. Fixed-force ensembleb. Molecule can be twistedc. 0.01pN <f<20pN can be measured

Drawbacks of MT:a. Difficult to make fast DNA

extension measurementb. Difficult to apply large forces

SmallmagnetDNA

Paramagneticparticle

micropipette

objective

Nonmagnetic bead

Vertical magnetic tweezer Transverse magnetic tweezer

a. Low noise long time measurementb. 0.01pN <f<20pN can be measuredc. Difficult to make fast real time

measurement

a. DNA is pulled in focus plane, real time measurement of extension

b. Magnet can be placed close to the magnetic bead, 0.1 pN <f <200pN

PRE,70, 011905 (2004)

Examples of real biophysics researchRNA synthesis

DNA mechanics Chromatins and chromosomes

Cell shapes

Backtracking by single RNA polymerase moleculesBlock’s lab at Stanford

Nature, 426, 684 (2003)

Transcription pauses:Short & frequent ones(several secs)Long pauses: [15s, 30minutes]

Backtracking and recovery associated with long pauses

No backtracking and recovery in short pauses

DNA micromechanics

A

Force-extension curves

ˆ 220

ˆˆ[ . ( ) ]tL

dAdsz tE ds fβ β= − +∫

A = 50 nm

DNA is Semi-flexible polymer at low force 65 pN leads to a structural transition

Thin but long DNA must be efficiently packed into micron-size cells.

Proteins able to fold, bend, loop, cross link, and disentangle DNAs are involved in DNA packing and DNA processing.

Understanding the interactions between DNA and these DNA organizing proteins is critical to understand DNA organizations in cells.

My thesis focuses on the study of DNA-protein interactions using single-DNA manipulations.

In animal cells:

•Up to ~1 m length

• ~10 um cell size (1 um = 10-6 m)

Random coil size ~ 250 um!

DNA packagingPhysics: how the DNA is packaged?

Physics: how the DNA is organized in cells?

Chromatin Assembly Using Interphase Xenopus Egg Extracts

Yan Jie, Dunja Skoko, John F Marko, Department of Physics, UIC

Rebecca Heald, Tom Maresca, MCB, UC Berkeley

Submitted to MBC• Direct structural information about compaction of DNA into chromatin• Possibility of direct observation of processing of chromatin, e.g.

chromatin remodeling, or mitotic chromatin condensation• Approximating in vivo: High speed extracts from Xenopus eggs, diluted in

buffer, assembles nucleosomes onto naked DNA (Ladoux et al, PNAS2000, Bennink et al, NSB 2001)

3μm3 microns

f=1.5 pN,bare DNA f=1.5 pN, assembly

3 microns-ATP

Physics: how the DNA is organized in cells?

Clearer images of bead-on-a-string structure~1mM salt

Chromosome cut by 1-10 nM micrococcal nuclease

Time (sec)

Forc

e (n

N)

Michael G. Poirier et al, from Marko’s lab at UICPNAS 99, 15393 (2002)

Physics: how the DNA is organized in cells?

Physics: what determines the cell shape?how the cell membrane elasticity contributes?how the cell membrane permits transportation?

Human red blood cell image

Helfrich energy model

Ou-yang shape equation

Many other topics:•Protein folding I unfolding•RNA structure•Enzyme activity•DNA replication, gene expression•Nucleic acid transportation•Protein searching for its specific binding sites•Gene expression control•Genome correlation, gene network•Neuron physics•And many others

Janaky Narayanan PC 2267 / NUS / AY 2004-05 Sem 1 15

Summary of the Biological Unit (Cell) The basic unit of life is the cell which can perform metabolism, self-reproduction and mutability. It is an open system; it permanently exchanges substances with its environment. The atoms and the elementary particles such as electrons are the basic elements of matter, both nonliving and living. However, a haphazard heap of all the molecules or even the aggregates which constitute a cell is not living. Only when they are arranged in a very special way, they form a matter, which has life. Thus cell is the lowest form of organization of matter which we can call life. Cells occur in a wide variety of sizes (nerve cells can be longer than a meter), shapes and functions. The structural and functional features of cells are fundamental to the study of life. A common structural feature in all cells is the membrane. The membrane surrounding the cell is called the cytoplasmic membrane (or plasmalemma or plasma membrane). It forms a selective barrier which maintains the chemical integrity of the cell. This is done by passive and active transport processes across the membrane which is selective to the kind of molecules that enter or leave the cell and the rate of their movement. Membranes can form a basis on which rapid chemical transformations take place. A variety of enzyme systems is associated with, or can be integral part of membranes. These enzyme systems govern the transport of ions and/or molecules and the rate of various biological reactions. Membranes occur as electrical insulation around fibrous extensions (called axons) of some nerve cells. Such insulation is called the myelin sheath. Advances in technology of electron microscopy have established a major morphological distinction between two groups of cells. The majority of cells belong to the group called eukaryotes. Only bacteria and the blue-green algae belong to the other group, the prokaryotes. In the prokaryotic cell no well developed internal structure in the form of extensive particulate organelles can be seen. The eukaryotic cell shows a highly differentiated internal structure, with visible particulate organelles. This type of cell has a well-defined nucleus surrounded by nuclear membrane.

Janaky Narayanan PC 2267 / NUS / AY 2004-05 Sem 1 16

Eukaryotic cell

Eukaryotic Organelles: (Organelles are supramolecular functional units) The intervening medium between plasma membrane and nucleus is called the cytoplasm. The nuclear membrane often shows continuity with membrane systems in the extranuclear part of the cell. The material inside the nucleus is a very fine thread like substance called Chromatin. The chromatin consists of the nucleic acids, deoxyribonucleic acid (DNA) and the ribonucleic acid (RNA), a low molecular weight protein called histone, and some residual protein. The nucleus may contain denser bodies called nucleoli. The DNA contains all the information for the morphology and function of the cell. When the cell divides, the chromatin becomes visible as elongated structures called chromosomes. There are channel-or-sac forming (cisternal) structures and endoplasmic reticulum which extend to a variable degree from the nuclear membrane to the cytoplasmic membrane. These channels help in segregating and transporting products to other parts of the cell or to outside environment. Numerous little granules (20 -25nm diameter) called ribosomes surround this endoplasmic reticulum. Ribosomes are instrumental for the synthesis of proteins. Another set of membranes seen in cells is the Golgi apparatus. Specialized cells with secretory functions have a well-developed Golgi systems. Centriole is an organelle which is found more often in animal than plant cells, and is part of the cell’s locomotive system. They have cylindrical structure (300 -500 nm long and 150nm diameter) and are critical in determining the axis of cell division. Lysosomes are vesicle-type organelles with an outer limiting membrane. They are filled with lytic enzymes involved in the break down of cellular fragments and large molecules. They remove foreign bodies and cell structures that are no longer needed. Mitochondria, called “power plant” of the cell produce most of the convertible energy in the cell. They range in size from 0.2 to 7.0μm, vary in shape from spheres to elongated rods. Plant cells differ from animal cells in that they have a sturdy cell wall made of cellulose or cellulose like material surrounding the cytoplasmic membrane. Another structural feature, more characteristic of plant than animal cells is the large liquid filled cavity in the middle of the cell called the vacuole. Cells found in green parts of the plants, and in most unicellular algae contain chloroplasts, organelles which are the site of phototrophic energy conservation (light energy converted to chemical energy by a process called photosynthesis).