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Dr.

Yongheng Liang
College of Life Science
Nanjing Agricultural University
Nanjing 210095
Tel/Fax: 025-8439 6376
Homepage: http://lfc.njau.edu.cn/common.asp?d=25&m=3&i=47

Research interesting: Protein trafficking in eukaryotic cells


Course in-charge-of:
1. Introduction to Yeast Biology (6/8/2016, 9:00am-12:00pm)
2. Yeast experiments (6/8/2016, 2:00-5:00pm)

Yeast Biology

Whats yeast?

Saccharomyces cerevisiae
(budding yeast/
Bakers yeast )

Schizosaccharomyces pombe
(fission yeast)

Candida albicans, a human pathogen


Symptom
Candida albicans

Outline for this course


Part I.

Yeast Basics

Part II.

Methods and techniques for studying yeast

Part III.

Cellular structures of Yeast

Part IV.

Yeast growth and division

Part V.

Techniques developed from yeast

Part VI.

Simple Yeast Experiment you can


teach in your classroom

Part I.

Yeast Basics
Mitochondria

Vacuole
Nucleus

Cell wall

Vesicle

1.1 Yeast History

S. cerevisiae was first


established as a tool for genetic
studies in the 1930's and 40's nearly 100 years after Mendel
performed the first controlled
genetic experiments on pea
plants.
In 1996 finished sequencing the
genome of S. cerevisiae (the first
eukaryotic genome to be
completed!).
By the end of the 20th century, S. cerevisiae was (and still is!) widely accepted as
one of the most powerful model systems for the study of genetics and biochemistry.

1.2 Yeast as a model organism

Yeast (Saccharomyces cerevisiae)


l Control of cell cycle and cell
division
l Cell differentiation
l Protein secretion and membrane
biogenesis
l Function of the cytoskeleton
l Gene regulation and
chromosome structure
l Aging

Eduard Buchner

German, 1860 1917

the Nobel Prize in


Chemistry in 1907
Discovery of non-cellular fermentation from yeast

2001 Nobel Prize in Physiology and Medicine


For their discovery in cell cycle regulation

Leland H. Hartwell

R. Timothy (Tim) Hunt

Sir Paul M. Nurse

2013 Nobel Prize in Physiology and Medicine

for their discoveries of


machinery regulating
vesicle traffic,
a major transport system
in our cells

WT, 37C

sec1-1, 37C

1h
sec1-1, 24C

sec1-1, 37C

2h
Novick P, Schekman R: Secretion and cell-surface growth are blocked
in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc
Natl Acad Sci USA 1979; 76:1858-1862.

Why yeast can become a popular


model organism?
nOne of the qualities that make S. cerevisiae particularly suitable for
genetic research is that it is a unicellular microbe. Can be grown on
defined media giving the investigator complete control over environmental
parameters.
nwhen frozen under the appropriate conditions, yeast remain viable and
genetically stable for years, perhaps indefinitely.
nIts small size makes easy to care for no large, smelly cages to clean
and maintain.
nPerhaps more importantly, S. cerevisiae is eukaryotic (as all Fungi are)
and thus yeast cells share much in common with human cells, and that
corresponding genes can often complement each other, unlike singlecelled bacteria (also known as prokaryotes).
nYeast also have many well-characterized genetic markers; the yeast
genome contains relatively little "junk DNA" (repetitive sequences, introns,
etc.)
nFurthermore, S. cerevisiae can be maintained in the haploid or diploid
state, thereby expanding the possibilities for genetic research by making it
easier to study both recessive alleles and genes essential for life.

Biotechnologies involving yeast

Useful Yeast Web Resources

1.3 Yeast morphology and development

Yeast colony

Size and Shape of Yeast


Haploid cell

Diploid cell

Volume (m3)

70

120

Diameter (m)

5-6

S. cerevisiae cells are generally


oval in shape ranging from 5 to
10 m at the large diameter and
1 to 7 m at the small diameter.
Mean cell volumes are 70 or 120
m 3 for a haploid or a diploid
cell, respectively; cell size
increases with age.

Morphology of budding yeast


DIC

FM4-64

Merged

1.4 Yeast reproduction


Asexual production
1). Budding yeastdaughter cell buds from
mature mother cell and grows up till finally
detaches from mother cell.

Budding process for budding yeast

Budding video

2). Fission yeastlike bacteria, mother cells


stretch out and then do a binary division to
form two daughter cells.

Is asexual reproduction
a mitosis or an amitosis?

The chromosomes are distributed


by actin (green) & microtubule
(red) cytoskeletons in fission
yeast during mitosis (160x)

The chromosomes are shown in blue,


the mitotic spindle in green and the
centrosomes in red.

Part II.

Methods and techniques for


studying yeast

FIVE IS
2. Incubation

1. Inoculation

4. Inspection

5. Identification

3. Isolation

Growing yeast on plates


Growing: Inoculation and Incubation

Streak

Dilute and
pour plate

Incubator

Growing yeast in liquid medium

flask rack

test tube rack

rotor drum

Spoke Inoculating Manifold 1/10

Channel pipette

200
180+20=200

1/100

180+20=200

1/1000

180+20=200

1/10000

180+20=200

96-well plates

Light microscopy Inspection

PhC

DIC

Other microscopies

GFP-Snc1 pem for plasma


membrane and FM4-64 for
vacuole

Nikon E800 fluorescence microscopy

Confocal
microscopy

Elo3-GFP
endoplasmic reticulum.
Nile red staining
of lipid droplets in obese yeast

Tansmission
electron-microscopy
Leica Ultracut

JEM-1011

TEM of yeast mutant at 37C

Scanning electron-microscopy

JEOL SEM

SEM for
budding yeast

Dissection microscopy

The Zeiss Tetrad Advanced Yeast Dissection


Microscope

Yeast cells can form tetrade through meiosis

The steps for sequentially separating the cluster of


four ascospores approximately 5 mm apart on petri
dishes.

lSpore colonies derived from asci separated on the surface of a petri


dish (Left). The spore colonies were replica plated to a synthetic
medium lacking a nutrient (Right).
lThe 2:2 segregation of a heterozygous marker is revealed by the
growth pattern on the selective medium.

Application of mating and dissection

Part III.

Yeast cellular structures and


functions

Classes of molecules encountered in yeast.

Macromolecular
constituents of
yeast comprise
proteins,
glycoproteins,
polysaccharides,
polyphosphates,
lipids, and
nucleic acids

nLocalize or follow the movement of particular


proteins within yeast cells with green
fluorescent protein (GFP) or other fluorescent
proteins.
nUse structure-specific fluorescent dyes to
visualize proteins.
Structure-specific dyes for yeast cells

DIC

DAPI staining

ypt1ts

DIC

Live microscopy

DAPI

ypt1ts

Htb1-CFP

Yeast Cell Organelles and Compartments

-Cell wall

PeriplasmPMNucleus

Vacuole
ER-

Autophsgic
bodies

Transmissional Electron Microscopy

Scheme of organelles and compartments in a yeast cell

Thin section of a yeast cell


Cell wall
periplasm
Bud

Plasma membrane
Nucleus
Vacuole

ER

The origin and detailed structure of the rough


endoplasmic reticulum (RER)

yeast endoplasmic reticulum with GFP or ER Tracker

ER labeled with GFP

DIC

GFP-Pmp2

ER visualized with fluorescence dye, ER Tracker

Details of Golgi apparatus

GFP-Snc1 and Sec7-DsRed in different yeast cells

DIC

WT

mutant

GFPSnc1

Sec7DsRed

merged

Localize a protein with reference to known


Golgi markers

The transport process

Exocytosis and endocytosis in budding yeast


Ypt10
VAC
LE

Ypt7

Ypt51,52,53
Ypt11

Ypt6
Ypt1

ER

E
E

Ypt31,32

Sec4

secretory PM
vesicles

GOLGI

Ypt small GTPases are involved in vesicle trafficking in yeast

Defect in transport process

Endocytosis

Vacuoles

DIC

GFP

FM4-64

merged

WT

mutant

The most convenient way to see the


vacuole is staining it with a dye FM4-64.

Model for autophagy in budding yeast

Category of Autophagy

Mictophagy (Mitochondria)
Chlorophagy (Chlorophyll)
Pexophagy (Peroxisome)

ypt51 affects GFP-Atg8 localization and degradation

merged

GFP

FM4

DIC

WT

ypt51

ypt52

ypt53

ypt51GFP-Atg8
SD-N

WT

ypt51

GFP-Atg8 and RFP-Ape1 in ypt51 and ypt51ypt52 strains

DIC

WT

ypt51

ypt51
ypt52

Atg8

Ape1

merged

A model of cytoskeleton

like the freeways for


vesicle to move on

Staining of actin with Rhodamine

Part IV.

Yeast growth and division

Life cycle of budding yeast

Budding

Budding is the most common


mode of vegetative growth in
yeasts.
Yeast bud and bud scar

How to bud?

Localized weakening of the cell wall


Tension exerted by turgor pressure
Extrusion of cytoplasm into an area bounded by new cell wall
material.
The regulation of particular cell wall synthetic enzymes and
transport of specific bud plasma membrane receptors are
key steps in the emergence of a bud.

The number of bud scars left on the surface of a yeast


cell is a useful determinant of cellular age.

Examples of components important for budding

Mutant phenotypes in
budding
Wild-type haploid
an axial budding pattern

Chitin of bud scars can


be detected by
Calcoflour white.

Wild-type diploid

a bipolar budding pattern

ste20-delta diploid

bud7-delta diploid

Location of protein components of the spindle pole body

The spindle pole body, SPB duplication pathway

SPB duplication can be divided into three steps: (1) half-bridge


elongation and deposition of satellite material, (2) expansion of the
satellite into a duplication plaque and retraction of the half-bridge,
and (3) insertion of the duplication plaque into the nuclear
envelope and assembly of the inner plaque. Following completion
of SPB duplication, the bridge connecting the side-by-side SPBs is
severed, and SPBs move to opposite sides of the nuclear envelope
(4).

Spindle dynamics

Septins

Regulation of the yeast cell cycle

START

Discovery of CDC genes

1960s Leland Hartwell, 1970s Paul


Nurse identified cell division cycle
gene, CDC, from budding yeast and
fission yeast with temperature
sensitive mutants

Identification of cdc28

Identification of cdc2 from fission yeast

1980s Timothy Hunt discovered cyclin

35S-methionine

Cyclin in sea urchin

2001 Nobel Prize for Physiology and Medicine

Leland H. Hartwell

R. Timothy (Tim) Hunt

Sir Paul M. Nurse

Nutrient levels regulate cell cycle

Mitosis vs. Meiosis

a
a

For diploid cells, though vegetative growth (Mitosis) is the


major way of yeast reproduction, sexual reproduction
(Meiosis) is an alternative when nutrient supplies fall short.

Meiosis
uThe life cycle of S. cerevisiae
normally alternates between
diplophase and haplophase. Both
ploidies can exist as stable cultures.
uIn heterothallic strains, haploid
cells are of two mating types, a and
. Mating of a and cells results in
a/ diploids that can undergo
meiosis.
uThe four haploid products
resulting from meiosis of a diploid
cell are contained within the wall of
the mother cell (the ascus).
Digestion of the ascus and
separation of the spores by
micromanipulation yield the four
haploid meiotic products.

ascus

Life cycle of yeast

Mating and sporulation


A. Haploid yeast cells budding
B. Haploid cells forming
shmoos and zygotes
C. Zygote budding off diploid
D. Diploid budding
E. Diploid forming asci with
ascospores; freed haploid
spores

Meiosis and Chromosome change

Summary of yeast life cycle in budding yeast

2N

Overview on stress responses in yeast

Part V.

Techniques developed from yeast

Principle of the yeast two hybrid system

Y2H for Vps34 with Vps8, Vps9 and Ypt51


AD
BD

Vp

s8

Vp

s9

Yp

1
5
t

8
s
p

9
s
p

Vps34
-Leu-Trp

-Leu-Trp-His

Yp

1
5
t

Summary for this course


Part I.

Yeast Basics

Part II.

Methods and techniques for studying yeast

Part III.

Cellular structures of Yeast

Part IV.

Yeast growth and division

Part V.

Techniques developed from yeast

Part VI.

Simple Yeast Experiment you can


teach in your classroom

Eduard Buchner

German, 1860 1917

the Nobel Prize in


Chemistry in 1907
Discovery of non-cellular fermentation from yeast

Sugar Catabolism in Yeast

Metabolism in yeast under aerobic and anaerobic conditions

Part VI.

Simple Yeast Experiment you may


teach in your classroom

How can yeast produce CO2 / gas?


What do you need for doing this experiment?
Test tubes
Test tube racks
Marker pen
H2O
Yeast
Sugars (Glucose or sucrose)
Balloon
Incubator
1

Yeast
H2O

H2O

Cap each test tube with a balloon.


Incubate tubes at 30C. At different
time points, observe which tube will
produce Gas ?

Sugar
H2O

Yeast 0.5g
Sugar 1g
H2O 5ml

Before

After

View yeast protein localization with fluorescence microscopy

l View Atg21 at the endosome in WT


l View Atg8 inside the vacuole in WT
PhC

WT

ypt51

Atg21-GFP

RFP-Atg8

merged

Grouping for afternoon experiment


Time

2:00-3:30

3:30-5:00

Room
Biology (20)

Chemistry (20)

C5023
for yeast
balloon exp
C6012

C6012
for fluorescence
observation
C5023

Ypt

ole
Vacu

Thank

You!

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