We’re finishing up the sea star ISH this week! This experiment has been going pretty smoothly, other than having to track down some emergency sheep serum today. We’ve added some abalone slides to the mix too, so we have 18 in situ hybridizations in total for counter staining and visualizing under the microscope tomorrow.
The real fun today was dicing up the cell adhesion story hidden in the Pycnopodia helianthoides transcriptome. I had to do a lot of reading and searching to really start understanding focal adhesion complexes and how they involve different proteins and downstream processes, so I started building that database of papers and stuck them in my folder on Google Drive.
I also wanted to better understand which focal adhesion genes change expression, and how the coelomocytes are responding to disease. It makes intuitive sense that focal adhesion might play a role in the clotting response we’re seeing from changes in the coagulation pathway. Let me paint a picture: fibronectin is an abundantly soluble protein in the extracellular matrix (ECM), although not physiologically active until self-assembled into fibrils. It binds to integrin receptors on the cell surface as well as extracellular matrix components such as collagen, fibrin, and heparan sulfate proteoglycans (Pancov 2002). Understanding the backdrop that the extracellular fibronectin matrix scaffolding plays in cell adhesion is important, but the interesting changes in gene expression are also at the cell-surface level: what focal adhesion receptor and substrate genes are changing and what signal pathways might be affected?
Integrins are the transmembrane proteins that make up an important part of the focal adhesion complex on the cell membrane, binding the cell to the ECM and setting off a potential inside-out signaling event between cell and ECM (Widmaier et al 2012). There’s a plethora of proteins that can interact at the focal adhesion junction.
The following table is a list of interacting proteins I pulled from Plow et al, 2000:
|Adenovirus penton base protein||αvβ3, αvβ5|
|Bone sialoprotein||αvβ3, αvβ5|
|Collagens||α1β1, α2β1, α11β1, αIbβ3|
|Denatured collagen||α5β1, αvβ3, αIIbβ3|
|Cytotactin/tenascin-C||α8β1, α9β1, αvβ3, αvβ6|
|Fibronectin||α2β1, α3β1, α4β1, α4β7, α5β1, α8β1, αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, αIIbβ3|
|Fibrinogen||α5β1, αMβ2, αvβ3, αxβ2, αIIbβ3|
|HIV Tat protein||αvβ3, αvβ5|
|Invasin||α3β1, α4β1, α5β1, α6β1|
|Laminin||α1β1, α2β1, α6β1, α7β1, α6β4, αvβ3|
|Neutrophil inhibitory factor||αMβ2|
|Thrombospondin||α3β1, αvβ3, αIIbβ3|
|Vitronectin||αvβ1, αvβ3, αvβ5, αIIbβ3|
|von Willebrand factor||αvβ3, αIIbβ3|
So, that’s cool. You might recognize some proteins in that list. Why are integrins and focal adhesion complexes important?
We see transcriptome-wide changes in at least 24 integrins or integrin-binding genes:
|Protein names||log2Fold Change||pvalue||Entry||Length (aa)|
|Disintegrin and metalloproteinase domain-containing protein 10 (ADAM 10)||2.77||1.42E-07||Q8JIY1||749|
|A disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAM-TS 3)||3.77||6.09E-13||O15072||1205|
|Disintegrin and metalloproteinase domain-containing protein 12 (ADAM 12)||4.17||3.68E-14||Q61824||903|
|A disintegrin and metalloproteinase with thrombospondin motifs 6 (ADAM-TS 6)||-2.33||0.002229||Q9UKP5||1117|
|A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAM-TS 13) (vWF-cleaving protease)||7.96||1.97E-16||Q76LX8||1427|
|Sushi/vWF, EGF and pentraxin domain-containing protein 1 (Polydom)||2.20||0.00026||A2AVA0||3567|
|Sushi/vWF, EGF and pentraxin domain-containing protein 1 (Polydom)||4.20||1.75E-06||A2AVA0||3567|
|Sushi/vWF, EGF and pentraxin domain-containing protein 1 (Polydom)||1.61||1.26E-05||A2AVA0||3567|
|Sushi/vWF, EGF and pentraxin domain-containing protein 1 (Polydom)||-1.91||0.005433||A2AVA0||3567|
|Integrin alpha-4 (CD antigen CD49d)||3.07||6.60E-09||Q00651||1039|
|Integrin alpha-4 (CD antigen CD49d)||3.63||6.34E-06||Q00651||1039|
|Integrin beta-1 (Fibronectin receptor subunit beta) (CD antigen CD29)||-1.40||0.001249||B0FYY4||798|
|Matrix metalloproteinase-19 (MMP-19)||4.22||3.85E-06||Q9JHI0||527|
|Matrix metalloproteinase-24 (MMP-24)||2.24||0.000174||Q9R0S2||618|
|Collagenase 3 (MMP-13)||3.39||2.10E-05||O77656||471|
|Collagenase 3 (MMP-13)||10.84||3.86E-43||O77656||471|
|Collagenase 3 (MMP-13)||7.82||1.48E-18||O77656||471|
|Matrix metalloproteinase-17 (MMP-17)||4.08||8.08E-09||Q9ULZ9||603|
|Matrix metalloproteinase-16 (MMP-16)||3.43||7.61E-07||Q9WTR0||607|
A lot of these proteins pop up as important clotting or anticlotting responses, especially sushi, ADAM proteins, and MMPs. It may be reasonable to think more in depth about the role that changes in focal adhesion have on signaling events. How are the cells, specifically the coelomocytes, responding to this infection?
Side note! In my search today I came across a really cool protein called integrin-linked kinase (ILK). In addition to being an awesome part of the focal adhesion complex, it’s a biochemically inactive kinase…. it doesn’t phosphorylate proteins! Here’s a phylogenetic tree I generated in String with ILK (first, left-most column of green and orange) and its known interacting proteins.