BB 681

Biological Physics at Microscopic Scales

LECTURE 5 + LECTURE 6

Ambarish Kunwar

Department of Biosciences and Bioengineering

IIT Bombay

akunwar@iitb.ac.in
http://www.bio.iitb.ac.in/~akunwar/
 Studying Cell: A Reductionist Approach
Reductionism
complex system – break it down – reduce the complexity
 Instructions for assembly
All man-made systems
have an assembly manual

Which is the most

complex man-made
system?

How to define and

measure complexity?

www.brobousa.com/images/Brobo_Manual_Dual_Vise_Assembly.jpg
 Instructions for assembly?

2.5 cm

Pygmy sea horse A mushroom Moon jelly Aurelia

(Figure 1.3) (Figure 1.15) (Figure 42.2)

(i) Parts / components, (ii) How many of each of these, (iii) Sequence of assembly
 Towards getting a component list for cells…

• Our quest for “assembly manual” starts from the study of cells
• Two questions to answer upfront:
1. There are so many types of organisms, how to study all of them? Or,
is it necessary to study all of them?
2. Most cells are small in size and not visible to naked eyes: how to
study objects that cannot be seen?
 Choice of experimental system
Molecular logic of life is highly conserved

5 µm

Cross section
of a cilium

15 µm

Cilia of Cilia of
Paramecium windpipe cells

Figure 1.16 0.1 µm

 Component list for cells…

• Our quest for “assembly manual” starts from the study of cells
• Two questions to answer upfront:
1. There are so many types of organisms, how to study all of them? Or,
is it necessary to study all of them?
It suffices to study few model systems which are easy to work with in
the laboratory (ease of growing, short generation time, etc.)
2. Most cells are small in size and not visible to naked eyes: how to
study objects that cannot be seen?

Escherichia coli
www.cdc.gov
Some strains inhabit our gut
Some strains are pathogenic
Saccharomyces cerevisiae
Baker’s yeast, wikipedia
Model systems
Zebra fish (Danio rerio) Caenorhabditis elegans
wikipedia A free living round worm
wikipedia Arabidopsis thaliana
Tropical fresh water fish
A weed (www.esa.int)
Neurospora crassa
Drosophila melanogaster Xenopus laevis
Bread mold (fungus)
Fruit fly, wikipedia African clawed frog, wikipedia
www.devbio.biology.
gatech.edu
 Reducing the complexity of a system
Break the system, or simplify it, to understand how it works…
Reductionism is the approach wherein the complexity of a
system is reduced to understand how the system works

www.wisdomtimes.com/wp-content/uploads/2012/04/destructive-behaviour.jpg
 Inputs from other fields
Two examples: centrifuge and microscopy
Biologists
Contributions from
Specific assays - markers
Physics, Chemistry,
Instrumentation engineering
• Table-top centrifuges
• High speed centrifuges
• Ultracentrifuge
Possible to go up to 200,000×g
Light microscopy
Scanning electron microscopy
Fluorescence microscopy
Concept 6.1 Transmission electron microscopy
 Exploring microscopy
All are variants of light
microscopy except SEM
Brightfield
50 µm
Brightfield Phase-contrast Differential-interference- and TEM
(unstained (stained specimen) contrast (Nomarski)
specimen)

Light microscopy allows

imaging of a live cell

50 µm
Confocal (without) Confocal (with)

10 µm
Fluorescence
10 µm
SEM/TEM: dead cells
Figure 6.3 Deconvolution

Small print: artifacts

introduced while preparing
specimens
1 µm

Super-resolution Super-resolution Scanning Transmission

(without) (with) electron 2 µm electron 2 µm
microscopy (SEM) microscopy (TEM)
 Cell fractionation

Centrifugation: separate Tissue Homogenization

cells Homogenate
various components based
on their density / size Centrifugation

1,000 g Supernatant poured into next tube

Microscopy: identify the 10 min
components in each pellet 20,000 g Figure 6.4
20 min

Biochemical methods: 80,000 g

determine metabolic Pellet rich in
nuclei and
60 min

function of each fraction or cellular debris 150,000 g

pellet Pellet rich in
3h
mitochondria
and chloroplasts

Differential Pellet rich in Pellet rich in

centrifugation “microsomes” ribosomes
The three domains of life: consist of Prokaryotes and Eukaryotes
Bacteria: Prokaryotes, uni-cellular Archaea: Prokaryotes, uni-cellular

2 µm
2 µm
Eukarya: multi-cellular
Kingdom Animalia

100 µm

Kingdom Plantae

Protists

Kingdom Fungi
 Eukaryotic Cell

• The basic structural and functional unit of every organism is one of two
types of cells: prokaryotic or eukaryotic

• Only organisms of the domains Bacteria and Archaea consist of

prokaryotic cells

• Protists, fungi, animals, and plants all consist of eukaryotic cells

A cut-away view of a generalized Eukaryotic Cell (Animal)

ENDOPLASMIC RETICULUM (ER)

Nuclear
RoughSmooth envelope
Flagellum ER ER NucleolusNUCLEUS
Chromatin
Centrosome
Plasma
membrane
CYTOSKELETON:
Microfilaments
Intermediate filaments
Microtubules
Ribosomes

Microvilli
Golgi apparatus
Peroxisome
Mitochondrion Lysosome

Figure 6.8a
A cut-away view of a generalized Eukaryotic (Animal cell)
Nuclear
envelope
Rough ER Smooth ER
Nucleolus NUCLEUS
Flagellum
Chromatin
Centrosome
Plasma
membrane

CYTOSKELETON:
Microfilaments
Intermediate filaments
Microtubules
Ribosomes

Microvilli
Golgi apparatus

Peroxisome
Lysosome Figure 6.8
Mitochondrion
A cut-away view of a generalized Eukaryotic Cell (Plant)
Nuclear Rough
envelope endoplasmic Smooth
NUCLEUS Nucleolus reticulum endoplasmic
Chromatin reticulum

Ribosomes
Central vacuole
Golgi
apparatus Microfilaments
Intermediate CYTOSKELETON
filaments
Microtubules

Mitochondrion
Peroxisome
Plasma membrane Chloroplast

Cell wall Plasmodesmata

Wall of adjacent cell

Figure 6.8c
A cut-away view of a generalized Prokaryotic Cell
Fimbriae

Nucleoid

Ribosomes

Plasma membrane

Cell wall
Bacterial
Capsule
chromosome
0.5 µm
Flagella
(a) A typical (b) A thin section through
rod-shaped the bacterium Bacillus
bacterium coagulans (TEM)
Structures present only
in some organisms are
labeled in red
Figure 6.5
 Comparing Eukaryotic and Prokaryotic Cells
• Basic features of all cells
-Plasma membrane
-Semifluid substance called cytosol
-Chromosomes (carry genes)
-Ribosomes (make proteins)

Prokaryotic cells are characterized by having

No nucleus
DNA in an unbound region called the nucleoid
No membrane-bound organelles
Cytoplasm bound by the plasma membrane

Eukaryotic cells are characterized by having

DNA in a nucleus that is bounded by a membranous nuclear
Eukaryotic cells are generally much
envelope
larger than prokaryotic cells
Membrane-bound organelles
Cytoplasm between the plasma membrane and nucleus
 Plasma membrane

• The plasma membrane is a

selective barrier that allows
sufficient passage of oxygen,
nutrients, and waste to service
the volume of every cell

• The general structure of a

biological membrane is a double
layer of phospholipids
 Nucleus
• The nucleus contains most of the DNA
in a eukaryotic cell

• Ribosomes use the information from

the DNA to make proteins

• The nuclear envelope encloses the

nucleus, separating it from the
cytoplasm

• The nuclear membrane is a double

membrane; each membrane consists
of a lipid bilayer
 Nucleus: DNA, Chromatin, Chromosome
• In the nucleus, DNA is organized into discrete units called chromosomes

• Each chromosome is composed of a single DNA molecule associated with

proteins

• The DNA and proteins of chromosomes are together called chromatin

• Chromatin condenses to form discrete chromosomes as a cell prepares to

divide
Endoplasmic reticulum – biosynthetic factory
Biosynthesis

Figure 6.11

Smooth ER

Rough ER Nuclear Smooth ER Rough ER

envelope

ER lumen
Cisternae
Ribosomes Transitional
ER
Transport vesicle 0.20 μm
 Endoplasmic reticulum – biosynthetic factory
• The smooth ER
• Synthesizes lipids
• Metabolizes carbohydrates
• Detoxifies drugs and poisons
• Stores calcium ions
The rough ER
• Has bound ribosomes, which
secrete glycoproteins
(proteins covalently bonded
to carbohydrates)
• Distributes transport
vesicles, proteins surrounded
by membranes
 Golgi apparatus – receiving and shipping
Golgi
apparatus Figure 6.12

cis face
(“receiving” side of 0.1 μm
Golgi apparatus) 1 Cisternae
2
3 4
6
5

trans face
(“shipping” side of TEM of Golgi apparatus
Golgi apparatus)
Golgi apparatus – receiving and shipping
 Ribosome - Protein Factories

Ribosomes are particles made of

ribosomal RNA and protein

Ribosomes carry out protein synthesis in

two locations
In the cytosol (free ribosomes)
On the outside of the endoplasmic
reticulum or the nuclear envelope
(bound ribosomes)
 Lysosomes – digestive compartments
Figure 6.13
Vesicle containing
Nucleus 1 μm two damaged 1 μm
organelles

Mitochondrion
fragment
Peroxisome
fragment
Lysosome
Digestive Lysosome
enzymes Lysosome

Plasma Peroxisome
membrane
Digestion
Food Mitochondrion Digestion
vacuole
Vesicle
(a) Phagocytosis: lysosome digesting food (b) Autophagy: lysosome breaking down
damaged organelles
 Dismantling and scavenging stolen cars
Stolen cars are taken to a workshop where they are dismantled…
Usable parts are sent to spare parts dealers
Unusable parts are sent to scrap dealers

www.team-bhp.com/forum/indian-car-scene/176568-police-unearth-workshop-stolen-cars-delhi.html
 Vacuoles - Diverse Maintenance Compartments

• A plant cell or fungal cell may have one or several vacuoles

• Food vacuoles are formed by phagocytosis

• Contractile vacuoles, found in many freshwater protists, pump excess water out of cells

• Central vacuoles, found in many mature plant cells, hold organic compounds and water
 Mitochondria and chloroplasts - Energy Converters

•Mitochondria are the

sites of cellular respiration,
a metabolic process that
uses oxygen to generate
ATP

•Chloroplasts, found in
plants and algae, are the
sites of photosynthesis
 Peroxisomes-Oxidation
• Peroxisomes contain enzymes that remove hydrogen
atoms from various substrates and transfer them to
oxygen (O2), thus producing hydrogen peroxide
(H2O2) as a by-product
• Some peroxisomes use oxygen to break fatty acids
down into smaller molecules that are transported to
mitochondria and used as fuel for cellular
respiration.
• Peroxisomes in the liver detoxify alcohol and other
harmful compounds by transferring hydrogen from
the poisons to oxygen.
• The H2O2 formed by peroxisomes is itself toxic, but
the organelle also contains an enzyme that converts
H2O2 to water.
 Cytoskeleton
Cytoskeletal network of fibers in eukaryotes
• Organizes the structure and activities of cell
• Extends throughout the cytoplasm Image acquired by
• Anchors sub-cellular organelles immunofluorescence
• Composed of three types of molecular assemblies spectroscopy
Microtubules, microfilaments, intermediate filaments Figure 6.20

microtubules: tagged green

10 μm
microfilaments: tagged orange
intermediate filaments: not tagged
DNA in the nucleus: tagged blue
Microtubules

Hollow tubes

Polymers
Elongate / shrink easily Table 6.1

Maintain cell shape (resist compression)

Cell motility (cilia/flagella)
Chromosome movement
Organelle movement
Cells used: fibroblasts
microtubules: tagged green
DNA in the nucleus: tagged blue
Microfilaments

Two intertwined strands

Table 6.1
Maintain cell shape (tension bearing)
Muscle contraction
Cytoplasmic streaming (plants)
Cell motility (amoeboid movement)
Cell division (animal cells)

Cells used: fibroblasts

microfilaments: tagged orange
 Intermediate filaments

Fibrous proteins – form cable-like coils

Table 6.1
Maintain cell shape (tension bearing)
Anchors nucleus, a few other organelles
Cytoplasmic streaming (plants)
Forms nuclear lamina

Cells used: fibroblasts

intermediate filaments: not tagged
Lamina: a mesh or network
DNA in the nucleus: tagged orange
 Motor proteins
Figure 6.21
Vesicle
ATP
Receptor for
motor protein
Motor proteins “walk”
vesicles along
Motor protein Microtubule cytoskeletal fibers.
(ATP powered) of cytoskeleton

Microtubule Vesicles 0.25 μm

Scanning electron micrograph of
a squid giant axon

Axon: is the extension of a nerve

cell (or neuron)

Shown are two

neurotransmitter-containing
vesicles moving towards the tip
of the axon
Cytoskeletal Filaments
Table 6.1

10
10 µm 10 5
5 µm
µm 10 µm
µm µm

Column
Column of
of tubulin
tubulin dimers
dimers
Keratin
Keratin proteins
proteins
Actin
Actin subunit Fibrous
subunit Fibrous subunit
subunit (keratins
(keratins
25 nm
25 nm coiled together)
coiled together)
7 nm 8−12 nm
7 nm 8−12 nm
α β
β Tubulin
Tubulin dimer
dimer
α
 Centrosomes and Centrioles
Centrosomes and CentriolesIn many
cells, microtubules grow out from a
centrosome near the nucleus

• The centrosome is a “microtubule-

organizing center”

• In animal cells, the centrosome has a

pair of centrioles, each with nine
triplets of microtubules arranged in a
ring
 Cilia and Flagella

• Microtubules control the

beating of cilia and flagella,
locomotor appendages of some
cells

• Cilia and flagella differ in their

beating patterns
 Cilia and Flagella

• Cilia and flagella share a common

structure
• A core of microtubules sheathed by
the plasma membrane

• A basal body that anchors the

cilium or flagellum

• A motor protein called dynein,

which drives the bending
movements of a cilium or flagellum
 Microfilaments (Actin Filaments)

• Microfilaments are solid rods about 7 nm in diameter, built as a twisted

double chain of actin subunits

• The structural role of microfilaments is to bear tension, resisting pulling

forces within the cell

• They form a 3-D network called the cortex just inside the plasma
membrane to help support the cell’s shape

• Bundles of microfilaments make up the core of microvilli of intestinal

cells
 Microfilaments (Actin Filaments)

• Microfilaments that function in cellular motility contain the protein

myosin in addition to actin

• In muscle cells, thousands of actin filaments are arranged parallel to one

another
 Microfilaments (Actin Filaments)
• Localized contraction brought about by actin and myosin also drives
amoeboid movement

• Pseudopodia (cellular extensions) extend and contract through the

reversible assembly and contraction of actin subunits into microfilaments
 Microfilaments (Actin Filaments)

• Cytoplasmic streaming is a circular flow of cytoplasm within cells

• This streaming speeds distribution of materials within the cell

• In plant cells, actin-myosin interactions drive cytoplasmic streaming
 Intermediate Filaments

• Intermediate filaments range in diameter from 8–12 nanometers, larger

than microfilaments but smaller than microtubules

• They support cell shape and fix organelles in place

• Intermediate filaments are more permanent cytoskeleton fixtures than

the other two classes
 Extracellular Compoments

Extracellular components and connections between cells help coordinate

cellular activities

• Most cells synthesize and secrete materials that are external to the
plasma membrane

• These extracellular structures include

• Cell walls of plants
• The extracellular matrix (ECM) of animal cells
• Intercellular junctions
 Cell Wall of Plants
• The cell wall is an extracellular
structure that distinguishes plant
cells from animal cells
• Prokaryotes, fungi, and some
protists also have cell walls
• The cell wall protects the plant
cell, maintains its shape, and
prevents excessive uptake of
water
• Plant cell walls are made of
cellulose fibers embedded in
other polysaccharides and protein
 The Extracellular Matrix (ECM) of Animal Cells

• Animal cells lack cell walls but

are covered by an elaborate
extracellular matrix (ECM)

• The ECM is made up of

glycoproteins such as collagen,
proteoglycans, and fibronectin

• ECM proteins bind to receptor

proteins in the plasma Functions of the ECM: Support, Adhesion, Movement, Regulation

membrane called integrins

 Cell Junctions
• Neighboring cells in tissues, organs, or organ systems often adhere,
interact, and communicate through direct physical contact

• Intercellular junctions facilitate this contact

• There are several types of intercellular junctions

• Plasmodesmata
• Tight junctions
• Desmosomes
• Gap junctions
 Plasmodesmata in Plant Cells

• Plasmodesmata are channels

that perforate plant cell walls

• Through plasmodesmata,
water and small solutes (and
sometimes proteins and RNA)
can pass from cell to cell