Scholars BIO 315
Instructor: Dr. Rebecca Kellum (THM 319 office)
Phone: 257-9741/e-mail: rkellum@uky.edu
Office Hours: anytime by appointment
Lecture: in THM 116, T and R, 2:00-3:15 pm
Lab: in THM B03, Section 001: M 9:00-11:50 am
Section 002: M 1:00-3:50 pm
Lab TA: Brandon Franklin brandon.franklin@uky.edu
Required Text: Essential Cell Biology, 4th edition (2014)
Alberts, et al. Garland Science
Canvas: Old exams, Echo recordings of lectures, lab
exercises, pdf files of lecture slides and discussion
articles, on-line homework assignments, and grade
postings available on Canvas.
Music lovers:
remind you
of something?
The scholars section are designed to give you
more stimulation….not necessarily more rigor.
Therefore, the primary differentiating feature of the scholars
sections is a set of research articles for discussion.
You will be asked to answer questions about the articles
in class discussions and will also be assigned essay format
questions on them to prepare you for similar questions
on in-class exams (either lecture or lab exam).
I will keep a record of your contributions. You must have
participation in 4 out of 6 discussion periods for full credit.
(Don’t worry. I’ll make sure you all do this.)
Grade Breakdown
Lecture 70%; Discussion 15%; Lab 15%
Lecture:
Lecture Exams: 56.5%
Homework: 17.5%
Discussion:
Discussion Participation: 7.5%
Article Questions: 7.5%
Lab:
Lab Reports: 7.5%
Lab Exams: 7.5%
Questions When Reading ANY Research Article:
Intro: What was known before the work described in the
paper? What new question is the paper addressing?
Materials & Methods: Were any new materials or
methods developed specifically for this study? Why
were they needed?
Results: What question is being addressed in each
figure, what experiment was used to answer that
question, and how were the results interpreted?
Discussion: What did we learn from the study? Are there
any caveats to the conclusions? What new or remaining
questions are there?
Also Posted on Canvas (Discussion Articles link)
On-line Homework on Canvas
10 assignments
All but one (HW7) due on Sun. at 11:59 pm.
-Feedback given to incorrect responses,
but not until entire assignment completed.
This is a non-ideal feature of Canvas.
-I recommend copying and pasting the entire
assignment to a Word document and beginning
work on it before the due date. Questions
appear in the order material is covered in class.
-You will get 3 attempts to answer each question
correctly. But you will receive a grade that is an
average of all attempts to minimize gaming of
the system. Late assignments will receive half credit.
-Article questions will also be managed on Canvas.
Note on Class Attendance
Class attendance will be taken sporadically to
encourage class participation that will be
beneficial to you.
This will not directly impact your course grade,
but will factor into my assessment of your
suitability for any post-graduate education.
My recommendation letter can only be as good
as the data you give me to use. An important
piece of data for any recommendation letter is
active and regular class participation. Exceptional
participation with a B grade often gets a better
recommendation than poor participation with an
A grade.
Alberts • Bray • Hopkin • Johnson • Lewis • Raff • Roberts • Walter
Essential
Cell Biology
FOURTH EDITION
Chapter 1
Cells: The Fundamental Units of Life
Copyright © Garland Science 2014
Cells come in a variety of shapes and sizes
mammalian nerve cell
Fig. 1-1
Paramecium
Chlamydomonas
budding yeast
Heliobacter pylori
Cells form tissues in multi-cellular plants and animals.
Fig. 1-5
plant root tip
animal kidney tubules
The invention of the
light microscope in the
mid-17th century allowed
discovery of cells by
Robert Hooke and
Antoni van Leeuwenhoek.
Two centuries later,
cells were proposed to
form the basis of all life.
Cells originate from other
cells only- Louis Pasteur
(1860)
Panel 1-1
THE LIGHT MICROSCOPE
-beam of light is focused on,
then diffracted off specimen
-diffracted light captured
and re-diffracted through pair
of glass lenses: 1) objective
lens and 2) ocular lens
(magnifying image up to
1000-fold)
-light from ocular lens
captured by lens of eye
Panel 1-1a
Cell visualized by
light microscopy
What are fibers inside
the cell made of?
Fluorescence microscopy
allows researchers to
see specific molecules
inside cells by labeling
them with fluorescent tags.
actin
Only light l
emitted from
fluorescent
tag allowed to
reach eye
have light filters
that are specific for
the fluorescent tag used
Excitation
Filter
Only light l that excites fluorescent
tag allowed to reach specimen
Emission
Filter
Panel 1-1b
Nuclear Division: microtubules of mitotic spindle and chromosomes
Methods for Labeling Molecules with Fluorescent Tags
*
1) specific binding of fluorescent stain-limited to only
a few molecules (e.g., DAPI-staining of DNA)
indirect labeling of protein with fluorescent tag
* 2) through
an antibody (Immunostaining)
3) in vitro labeling of protein with fluorescent tagthen inject into living cell
*
4) in vivo expression of the protein fused to a naturally
fluorescing protein (e.g., GFP-tagging)
in lab
Immunostaining requires an antibody
B Cells Produce Antibodies
Cell Biologists Can Raise
Antibodies to Specific Proteins
They are used in immunostaining
and other types of cell biology
experiments.
Panel 4-2b
variable
non-variable
Immunofluorescence
Anti-actin
Antibody
(Ab)
actin
fluorescently labeled
antibody used to
indirectly label protein
(actin here) in fixed cell
DAPI-staining
Usually the fluorescent tag is linked
to a 2o antibody that recognizes any
1o antibody from a given species.
Observed in
fixed cell
variable
non-variable
2o antibodies recognize the non-variable
portion of the 1o antibody.
in vitro-labeling
Fluorescent tag covalently
linked to protein in test tube
fluorescently labeled
protein (actin here)
injected into living cell
Labeled protein incorporates
into its normal cellular structure
Observe in
a living cell
Expression of GFP-tagged protein
Example:
for GFP-tagged actin expressed in a living cell
GFP Tagging
GFP-tagged skin protein in mice
Aequoria victoria
Green Fluorescent Protein from
jellyfish can be fused to any protein
and expressed in almost any cell type
GFP-tagged wing protein in fly
Method 3: GFP Tagging
actin
GFP coding sequence DNA:
coding sequence DNA:
ATG CCC CGT AAA…
ATG GGG AAG GGA CCG TTG….
Transcription
Translation
actin
Transcription
Translation
protein
GFP
protein
actin-GFP fused coding sequence DNA
ATG GGG AAG GGA CCG TTG CCC CGT AAA…
Transcription
Translation
actin-GFP
fusion protein
fusion gene inserted
into genome of cell and
protein then expressed
in living cell
will be used in several
of our discussion articles
All 3 Methods for Labeling Proteins Should Produce
the Same Result (Will Discuss Further in Lab)
Earliest Microscopes
of 1600’s
single biconvex lens
A. van Leeuwenhoek
(microscope hobbyist)
270x Mag
1.35 mm Res
double lens (compound)
R. Hooke
(Royal Society of Science)
Modern Microscope: 1500x Mag and 0.2 µm Res
Higher Resolution
and Magnification
Microscopes magnify and resolve.
Higher
Magnification
Only
Resolution = distance (d)
between two points that
can be discriminated
limited by wavelike properties
of light (λ) and light gathering a
ability of lens (N.A.)
d=
0.61 λ
n sin α
N.A.
α = angle of
cone of light
collected
Theoretical Resolution Limit: 0.2 mm
Fig. 18.3
New Super-resolution microscopes
push that limit (Article #2)
Electron Microscope
Light Microscope
Electron Microscopy Allows Higher Resolution
Uses an electron
beam instead of
light beam
(allows lower l)
Karp CMB7
ELECTRON MICROSCOPE
-beam of accelerated electrons
instead of beam of light focused
on specimen
-electromagnet lenses capture
and diffract electron beam to
magnify specimen up to
250,000-fold
-can resolve points 1 nm (10-9 m) apart
Gold-labeled antibodies can be
used to label molecules to provide
specificity along with higher resolution.
Panel 1-1f
Immunoelectron Microscopy of Membrane Proteins
occludin (large Gold, orange arrowhead)
claudin (small Gold, black arrowhead)
Karp CMB
Ch 5-8
Ch 11, 12
Ch 15
Ch 1-4 lay
foundation
Ch 13, 14
Ch 16-18
ties all
together
Ch 15
Ch 13, 14
Fig. 1-7
X-Ray Diffraction Gives Even Higher Resolution(level of individual atoms in molecules)
Myoglobin
Karp CMB7
Maximum Resolution ~ 1 Ao (10 -10 m)
(10 x higher than EM)
Article #1
Model Organisms Have Been Used to Learn About Cells
Both genetic and biochemical experimental tools available.
Drosophila
unicellular yeast
C. elegans
mice
Zebra fish
Arabidopsis
Human Genes
Can Substitute
for Mutant
Yeast Genes!
How We Know, pg. 30
Human Cells Can Also Be Cultured Outside of Body (in vitro)
fibroblast cell
(found in connective tissue)
Fig. 1-38
skeletal muscle cell
epithelial cell
(found lining tissues)