3. Hypothesis and Experiment Design
We supposed that the newly discovered cell polarity regulator MRCK-1 are
required for the timely internalization of mesodermal precursors through either
an PAR proteins dependent or independent mechanism.
Use protein localization and disruption of function experiments to test the role of
MRCK-1in the mesodermal precursors timely internalization .
If MRCK-1 influence timely internalization of mesodermal precursors through PAR
proteins dependent mechanism.
Use Colocalization and FRET experiments to testify the interaction between
MRCK-1 and PAR proteins.
4. Material and Methods
Embryo is an important model to study about
morphogenesis. In term of morphogenesis, 66 cells
internalize during C. elegans gastrulation. By genetically
manipulate the cell polarity and cytoskeletal regulators, we
can study about diverse mechanisms of cell internalization
within a single organism.
▶ Ease of operation.
- Small size and transparent.
- Short life cycle.
▶ Commonalities with other advanced-organisms.
- Eukaryotic and multicellular.
- The genome(~10 MB) is with 40% homology to human(3.2 GB).
- It has digestive system, brain and muscle.
- *Embryo to adult through cell differentiation and proliferation.
Why do we use C. elegans as models?
5. Material and Methods
▶ Acquire strains from Caenorhabditis
Genetics Center (CGC), University of
Minnesota, Minneapolis, MN.
▶ To maintain the strain, bacterial food
source (OP50) and *NGM petri plates are
important.
▶ C. elegans can best be maintained between
16°C and 25°C, most typically at 20°C.
C. elegans acquisition and maintenance
● Wild type (AF16)
● CEH-51::GFP (MS632)
● par-3::PAR-3;ZF1-GFP
● par6::PAR6-ZF1-GFP
http://gizmodo.com/10-of-the-years-most-beautiful-science-images-508969751
*NGM media contains peptone, MgSO4
, CaCl2
,
cholesterol etc.
6. Material and Methods
▶ Tools used. Shown are a test tube containing
melted Vaseline, a paint brush with tape
wrapped around it to hold it in place, an
aspirator with a glass needle attached at one
end, a Q-tip stick with an eyelash taped at its
end, a surgical blade to cut worms, a watch
glass in which the worms are cut, and glass
capillary tubes used to pull the glass needle
on the aspirator.
▶ Corpses of worms cut in half and the
embryos released.
▶ Embryos mixed with *polymer beads after
mounting.
Mounting C. elegans embryos
http://cshprotocols.cshlp.org/content/2011/9/pdb.prot065599/F1.large.jpg
https://www.ncbi.nlm.nih.gov/pubmed/21880814
*Polymer beads serve as spacers between the coverslip and the
glass slide to prevent the embryo from being crushed.
7. Sample preparation and immunostaining
Freeze-cracking: frozen embryos are incubated in -20°C MeOH for 5 minutes, then in
secondary fix (2% paraformaldehyde, 48 mM PIPES, 25 mM HEPES (pH 6.9), 10 mM EGTA (pH
7.5), 2 mM MgCl2) at room temperature for 10 minutes.
Immunostaining: incubate embryos with 1% BSA, and glycine in PBST for 30 min to block
unspecific binding of antibodies. Then, incubate embryos in the primary antibodies at 4 °C
overnight. Wash with PBS and incubate with secondary antibodies at 37 °C for 1h at room
temperature in the dark. Wash 3 times with PBS.
http://www.slideshare.net/InnovaBiosciences/an-intr
oduction-to-immunohistochemistry
Material and Methods
8. Microscopy technique
Material and Methods
Laser scanning confocal microscopy:
● Fluorescent
● Time-lapse DIC
● Colocalization
● FRET
http://itn-snal.net/2014/10/laser-scanning-confocal-microscopy-lscm/
9. Experiment 1: Localization
Purpose: To examine the endogenous MRCK-1 distribution in mesodermal lineage during
internalization using fluorescent microscopy.
Method: Immunostain MRCK1 with RFP fluorescent labels, using CEH-51::GFP to identify
MS descendants at the MS16 stage. Compare apical and basal localization of MRCK-1 at the
stage of MS cell internalization, and for comparison at the MS4 stage, when MRCK-1 is
known to be apically enriched.
Procedure:
● Embryo preparation: CEH-51::GFP embryo
● Freeze-cracking
● Immunostaining:
○ blocking serum→primary antibody→ secondary antibody
Protein Primary antibody Secondary antibody λex
/λem
MRCK-1 Mouse anti-MRCK1 Mouse anti-RFP 555nm/584nm
10. Localization
Data Acquisition: Laser Scanning Confocal Microscopy.
● Fluorescent mode
● FITC filter for GFP, TIRTC filter for RFP.
● Photomultiplier tubes(PMT) detector.
Data processing: Metamorph software measures fluorescent intensity. CEH-51::GFP
levels identifies the basolateral and apical membranes through its localization
borders. Apical and basolateral GFP pixel intensity fall-offs can be used to align
anti-MRCK-1 intensity measurements between embryos.
https://www.thermofisher.com/us/en/home/life-science/cell-analysis/la
beling-chemistry/fluorescence-spectraviewer.html
http://www.pooher.com/xinwen/Technical/2015-04-14/973.html
11. Localization
Expected results:
MRCK-1 might localize apically in MS lineage cells specifically when cells internalize, or it
functions early to establish polarity that will be used later.
Jessica et al ., 2011
12. Experiment 2: Disruption of MRCK-1 function
Purpose: To test if MRCK-1 will play a major role in the timely internalization of MS cells
by recording time-lapse DIC images.
Method: Use mrck-1 mutant embryo to test its timely internalization changes compared with
wild type, then decide whether it will greatly influnce this process. Use mrck-1 and par-3 or
par-6 double mutant embryos to test whether MRCK-1 depends on PAR proteins to influence
MS cells timely internalization.
Procedure:
Embryo preparation:wild type embryo, mrck-1 mutant embryo, , mrck-1& par-3 double
mutant embryo, mrck-1 & par-6 double mutant embryo
13. Data acquisition: Laser scanning confocal microscope
● Time-lapse DIC: Time-lapse images will be acquired every minute.
● Detector: Photomultiplier tubes(PMT)
Data processing: Timing of MS divisions and internalization in the MS lineages will be
recorded every 1 minute. Internalization will be scored when a cell was beneath the surface
of the embryo, fully covered by other cells, before division.
Disruption of MRCK-1 function
14. Disruption of MRCK-1 function
Expected results:
The timely internalization of MS cells significantly changes only in mrck-1 embryos
compared to wild type, or only in mrck-1&par-3 and mrck-1&par-6 embryos.
Jessica et al ., 2011
15. Experiment 3: Colocalization
Purpose: To test if there is potential interaction between MRCK-1 and PAR-3/PAR-6 by
using colocalization microscopy technique.
Method: Immunostain MRCK-1, PAR-3, and PAR-6 with three different fluorescent labels,
each has separate emission wavelengths, and to investigate if there is spatial overlap
between MRCK-1 and PAR-3/PAR-6.
http://edoc.hu-berlin.de/dissertationen/easwaran-hariharan-2003-10-01/HTML/chapter3.html
16. Colocalization
Procedure:
● Embryo preparation
● Freeze-cracking
● Double immunostaining: MRCK-1/PAR-3, MRCK-1/PAR-6
○ Sequential incubation: first blocking serum→first primary antibody→ first
secondary antibody→second blocking serum (serum from the species that
the secondary antibody was raised in)→second primary antibody→second
secondary antibody
Protein Primary antibody Secondary antibody λex
/λem
PAR-3 Rabbit anti-PAR-3 Rabbit anti-RFP 555nm/584nm
PAR-6 Rabbit anti-PAR-6 Rabbit anti-YFP 514nm/527nm
MRCK-1 Mouse anti-MRCK1 Mouse anti-GFP 488nm/510nm
17. Data acquisition: Laser scanning confocal microscope
● Objectives
● Band pass filter (RFP, GFP, and YFP)
● Detector: Photomultiplier tubes(PMT)
http://www.visitech.co.uk/assets/imaging-of-fluorescent-proteins.pdf
Colocalization
http://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html
19. Experiment 4: FRET
▶ Purpose: To quantitatively analyze if there is potential interaction between
MRCK-1 and PAR3/PAR6 applying FRET acceptor photobleaching technique.
▶ Method: Apply freeze-cracking and immunostaining technique to embryos,
label MRCK-1 with YFP(acceptor) and separately label PAR 3/6 with CFP
(donor).
Protein Primary antibody Secondary antibody λex
/λem
MRCK - 1 Anti - MRCK - 1 Goat - Anti - YFP 514nm/527nm
PAR - 3 Anti - PAR - 3 Rabbit anti - CFP 436nm/477nm
PAR - 6 Anti - PAR - 6 Rabbit - Anti - CFP 436nm/477nm
20. FRET - Acceptor Photobleaching
Data Acquisition:
▶ Laser Scanning Confocal Microscopy (Some of the
microcroscopy need filter set, depends).
▶ Photomultiplier tubes(PMT) detector..
▶ Filter Set for Acceptor- YFP and Donor - CFP.
▶ Image processing software: (e.g., IPLab Spectrum;
SignalAnalytics)
● Omega-prepackaged set(XCY-500)
● A GFP/YFP filter set consists of a 510/20 filter
for GFP detection, and a 550/30 filter for YFP
detection. a 525 SP (or LP) dichroic mirror to
split the signals.
● 488 nm used as the excitation wavelength.
http://photobiology.info/Visser-Rolinski.html
21. FRET - Acceptor Photobleaching
Expected Result:
● Absence of Acceptor molecule lead to
increase of signal of donor molecules.
http://www.nature.com/nprot/journal/v1/n2/fig_tab/nprot.2006.122_F6.html
http://cshprotocols.cshlp.org/content/2006/6/pdb.prot4598.long
Acceptor Donor
22. Conclusion
Based on our experiment designed, once we observe MRCK-1 apical
localization during MS cells timely internalization from localization
experiment, and identify significant changes of MS cells timely
internalization in mrck-1 & par-3 or mrck-1& par-6 mutant embryo
compared to wild type, we can get a preliminary conclusion that MRCK-1
plays an important role in the MS cells timely internalization through PAR
proteins dependent mechanism.
Further colocalization and FRET experiment testify whether there are
interactions between MRCK-1 and PAR proteins, once we get positive results
from these experiments, it will indicate that MRCK-1 directly interacts with
PAR-3 or PAR-6 proteins to influence MS cells timely internalization.
23. References
▶ Harrell, Jessica R., and Bob Goldstein. "Internalization of Multiple Cells during C. Elegans
Gastrulation Depends on Common Cytoskeletal Mechanisms but Different Cell Polarity and
Cell Fate Regulators." Developmental Biology. 350.1 (2011): 1-12.
▶ Marston, Daniel J., Christopher D. Higgins, Kimberly A. Peters, Timothy D. Cupp,
Daniel J. Dickinson, Ariel M. Pani, Regan P. Moore, Amanda H. Cox, Daniel P. Kiehart,
and Bob Goldstein. "MRCK-1 Drives Apical Constriction in C. elegans by Linking
Developmental Patterning to Force Generation." Current Biology 26.16 (2016): 2079-089.
▶ Bao, Z., and J. I. Murray. "Mounting Caenorhabditis Elegans Embryos for Live Imaging of
Embryogenesis." Cold Spring Harbor Protocols. 2011.9.
▶ Verveer, P. J., O. Rocks, A. G. Harpur, and P. I. H. Bastiaens. "Imaging Protein Interactions by
FRET Microscopy: FRET Measurements by Acceptor Photobleaching." Cold Spring Harbor
Protocols. 2006.6.
