The Role of MRCK-1 in the C. elegans mesodermal precursors timely internalization

Flora Zi Yang, Yiyi Yao and Yiwen Zhu

Background

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.

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?

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, CaCl

2,

cholesterol etc.

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.

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-introduction-to-immunohistochemistry

Material and Methods

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/

Experiment 1: LocalizationPurpose: 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

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/labeling-chemistry/fluorescence-spectraviewer.html http://www.pooher.com/xinwen/Technical/2015-04-14/973.html

LocalizationExpected 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

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

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

Disruption of MRCK-1 functionExpected 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

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

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 λ

ex/λ

em

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

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

ColocalizationExpected results: ● Co-occurrence vs. correlation

● Pearson's correlation coefficient and R2

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3074624/

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

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

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

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.

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.

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