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Antwi-Adjei et al. Vessel Plus 2021;5:35 https://dx.doi.org/10.20517/2574-1209.2021.48 Page 5 of 10

Figure 1. CCM3 is a novel regulator of a conserved Hippo-like signaling pathway. The fission yeast Morphogenesis (Mor) pathway is
defined by a short kinase cascade consisting of a Sterile20-like kinase (Nak1p) that phosphorylates and activates an NDR kinase
(Orb6p) which in turn phosphorylates effectors required for cell polarity and separation [48] . Many of the proteins in this pathway are
conserved among eukaryotes, with CCM3 representing a novel scaffolding protein, not present in yeast, for the upstream kinase
complex. CCM3 binds directly to GCKIII in human, zebrafish and flies [12,49,50] . Likewise, Mo25 can bind directly with Sterile20-like
kinases including GCKIII family members [51] . Direct interactions between CCM3 and Mo25 have yet to be reported.

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zone dilations. The penetrance of the dilation defects varied from allele to allele, with the fry allele
showing the most penetrant phenotype (~80%, 25/31 cells showing clear transition zone dilations). In
addition to transition zone tube dilations, less prominent tube dilations throughout the terminal cells were
also observed, consistent with our prior results for other pathway members. In addition, we observed gas-
filling defects and melanization of the transition zone dilation - these defects were less common, with lack of
gas-filling observed in about 5% of mutant cells and melanization in about 2% of mutant cells (n = 183 cells
scored). All of these defects were also described for tricornered mutant terminal cells , and none were
[21]
observed in wild type control larvae, or in the heterozygous terminal cells in fry mosaic larvae (n = 50 and
50, respectively). The incomplete penetrance of the fry phenotype may indicate a less stringent requirement
for fry than for GckIII and trc, or may reflect the presence of maternally supplied fry mRNA and/or protein.

DISCUSSION
The molecular mechanisms by which CCM3 acts in the vascular system remain unclear. Strong data
support a role of GCKIII family kinases acting together with CCM3, but the downstream target(s) of the
kinase have remained elusive. Our prior work in Drosophila suggests that the NDR kinases (Trc in flies,
[21]
STK38, STK38L in human) are likely to be the direct targets of the CCM3/GCKIII complex in endothelial
cells. Studies linking the scaffolding protein Furry to Trc function implied that Furry was also likely to be
required as part of the CCM3-GCKIII signaling cascade. Here we establish that at least in the fly tracheal
system, the Furry/Trc complex is regulated by CCM3/GCKIII. The connection between the CCM3-GCKIII
complex and a Trc-Fry-Mob complex was suggested by in vitro work using human orthologs [52,53] ; however,
other factors such as mTor or Hippo have also been proposed to act as upstream activators of Trc [32,33,40,54] .
Our data in the Drosophila wing demonstrate that at least in two tissues, wing and trachea, Fry/Trc are
regulated by the CCM3 pathway. Further studies in additional Trc-requiring tissues, such as neurons and
follicle cell epithelia, will need to be carried out determine whether the CCM3 pathway is a tissue-specific or
general regulator of Fry/Trc activity. Likewise, it will be critical to extend the analysis to the vertebrate
vascular system. If STK38 and STK38L are required downstream of CCM3 in endothelial cells, as expected,