A22 - Investigating the role of the membrane-associated Cerebral Cavernous Malformations (CCM) protein scaffold in biomechanical signaling

Principal Investigator

Prof. Dr. Salim Seyfried, Universität Potsdam

Correct vascular development and integrity depend on a complex of proteins called Cerebral Cavernous Malformation (CCM) proteins, which have inhibitory roles during angiogenesis and attenuate Rho kinase signaling, which limits vascular permeability 1, 2. A loss of the three intracellular proteins CCM1 (also known as Krev interaction trapped protein 1, KRIT1), CCM2 (also known as Malcavernin), or CCM3 (also known as programmed cell death protein 10, PDCD10) results in cerebral cavernous malformations 2, a group of vascular diseases characterized by cerebral hemorrhages that occur predominantly within low-flow venous capillary beds and that lack typical vessel wall components such as pericytes and vascular smooth muscle cells 3-5. Proteins of the CCM scaffold assemble around the transmembrane protein Heart of glass (HEG) 6. The entire CCM protein scaffold is involved in endothelial junctional stabilization and directly interacts with the vascular endothelial-cadherin (VE-cadherin) complex 7(Fig. 1A). In addition, the CCM protein scaffold represses the activity of the extracellular matrix-binding ß1 integrin by stabilizing KRIT1-binding Integrin cytoplasmic domain associated protein 1 (ICAP1) 8(Fig. 1A), a specific inhibitor of ß1 integrin9. A loss of CCM proteins causes a number of cardiovascular malformations in zebrafish including cardiac ballooning, heart looping defects, a failure of endocardial cushions to form, and defective blood vessel formation (Fig. 2)10.

Project A22 aims at elucidating the precise dynamics of CCM scaffold assembly and at identifying associated proteins during zebrafish endothelial development. In functional studies, using appropriate zebrafish mutants, we will address how the loss of AJs components impacts CCM protein scaffold assembly. Conversely, we will study the role of the CCM protein scaffold for AJs maturation within nascent blood vessels. Transgenic reporter lines for AJs are available for these analyses. In part, these studies will involve functional developmental genetics and cell biological studies. We will also put a strong emphasis on developing machine learning algorithms for advanced image analysis and work on improving superresolution microscopy techniques for in vivo applications in the zebrafish embryo.

 

Figure 1: Crosstalk of membrane-associated junctional complexes and signaling pathways with the CCM protein scaffold.
(A) The membrane-associated Heart of glass (HEG) protein scaffold, VE-cadherin (Cdh5)-based endothelial AJs, and integrin-based cell-extracellular matrix junctions play important roles in different steps of vessel morphogenesis. The two junctional complexes directly interact with the CCM protein scaffold via KRIT1/CCM1 or ICAP1. Direct interactions of KRIT1 and CCM3 with other proteins are indicated. CCM3 also interacts with kinases of the germinal center kinase III (GCK-III) family and with the striatin-interacting phosphatase and kinase (STRIPAK) complex (modified from 1). (B) Zebrafish Heart of glass is a single pass transmembrane protein containing a large extracellular domain with two epidermal growth factor-like (EGF-like) domains, a transmembrane segment and a short cytoplasmic tail with a conserved C-terminal NPXY/F. The extracellular domain is predicted to be highly glycosylated.

 

Figure 2: Cardiovascular defects in the zebrafish embryo associated with the loss of CCM2.
(A-D) Wild-type and (E-H) ccm2m201 mutant embryos. (A,E) Frontal views of 48 hours post fertilization (hpf) hearts of the myocardial reporter line Tg(myl7:GFP)twu34. (E) Cardiac expansion phenotype in ccm2m201 mutant. (B,F) Cardiac malformation phenotype in ccm2m201 mutants. (B’,F’) Shown are hearts of different genotypes at 48 hpf with a single confocal plane section at the atrioventricular canal region. Endocardial cushion cells are marked by Tg(kdrl:GFP)s843 and Alcam staining (asterisks). (F’) Complete lack of cardiac cushions in ccm2m201 mutant. (C,G) Dorsal views of the lateral dorsal aorta (LDA) at 48 hpf. The lateral dorsal aorta morphology is marked by Tg(kdrl:GFP)s843 expression (inverted image). (G) Overgrowth of the ccm2m201mutant lateral dorsal aorta. (D,H) Ectopic sprouts and vessel branch point defects within the subintestinal vein in ccm2m201mutants. A, atrium; V, ventricle

References:

  • Faurobert,E. & Albiges-Rizo,C. (2010) Recent insights into cerebral cavernous malformations: a complex jigsaw puzzle under    construction. FEBS J. 277, 1084-1096.

  • Fischer,A., Zalvide,J., Faurobert,E., Albiges-Rizo,C., & Tournier-Lasserve,E. (2013) Cerebral cavernous malformations: from CCM  genes to endothelial cell homeostasis. Trends Mol. Med. 19, 302-308.

  • Voss,K. et al. (2007) CCM3 interacts with CCM2 indicating common pathogenesis for cerebral cavernous malformations.  Neurogenetics.  8, 249-256.

  • Zawistowski,J.S. et al. (2005) CCM1 and CCM2 protein interactions in cell signaling: implications for cerebral cavernous malformations  pathogenesis. Hum. Mol. Genet. 14, 2521-2531.

  • Zhang,J., Rigamonti,D., Dietz,H.C., & Clatterbuck,R.E. (2007) Interaction between krit1 and malcavernin: implications for the  pathogenesis of cerebral cavernous malformations. Neurosurgery 60, 353-359.

  • Kleaveland,B. et al. (2009) Regulation of cardiovascular development and integrity by the heart of glass-cerebral cavernous  malformation protein pathway. Nat. Med. 15, 169-176.

  • Glading,A., Han,J., Stockton,R.A., & Ginsberg,M.H. (2007) KRIT-1/CCM1 is a Rap1 effector that regulates endothelial cell cell junctions.  J. Cell Biol. 179, 247-254.

  • Faurobert,E. et al. (2013) CCM1-ICAP-1 complex controls beta1 integrin-dependent endothelial contractility and fibronectin remodeling.    J. Cell Biol. 202, 545-561.

  • Millon-Fremillon,A. et al. (2008) Cell adaptive response to extracellular matrix density is controlled by ICAP-1-dependent beta1-integrin  affinity. J. Cell Biol. 180, 427-441.

  • Renz, M. et al. (2015) Regulation of β1 integrin-Klf2-mediated angiogenesis by CCM proteins.Dev. Cell 32, 181-90.