Page 65 - Q. Neuroscience

Mechanosensitive Piezo Channel, PEZO-1, regulates
intestinal motility in C. elegans

Yeon-Ji Park, Jihye Yeon and Kyuhyung Kim
Department of Brain and Cognitive Sciences, DGIST, Daegu, 42988, Korea

Abstract Mammalian two PIEZO genes, Piezo1 and Piezo2, have been shown to be involved in mechanosensation (Coste et al., 2010). C. elegans genome
has a single PIEZO gene, pezo-1, which encodes 14 isoforms and of which function has not been explored yet. The pezo-1 long isoforms are expressed in the pharyngeal-
intestinal valve cells, which appear to mediate the movement of food from the pharynx into the intestine (Avery et al.,1997). To identify roles of PEZO-1 in valve cells-
mediated food movement, we fed animals with GFP-microsphere and found that pezo-1 mutant animals show excess accumulation of GFP in the intestine lumen. Defects in
pezo-1mutants are restored by expression of a long isoform PEZO-1. In addition, ectopic expression of mouse PIEZO1 is sufficient to restore defects of pezo-1 mutants. Two
gap junction genes, inx-2 and inx-20, have been shown to connect the valve cells to the pharyngeal muscles. We found that inx-2 and inx-20 mutants also show defects in the
food movement. Currently, we are identifying the molecular mechanisms by which PEZO-1 and gap junction channels modulate valve cells-mediated food movement.
Furthermore, we are investigating whether PEZO-1 is activated by mechanical stimuli by performing electrophysiology in a heterologous system.
Introduction
mPIEZO1 can substitute PEZO-1 function in C. elegans
inx-2 and inx-20 mutants animals show defects
Piezo ion channel is activated by pressure in valve-cells mediated food movement
tm10725
- pezo-1p(L):: Wild-type inx-2;inx-20
Wild-type mPIEZO1 15,000
- Evolutionarily conserved gene from human to C. elegans 15,000 Line 1
; Piezo1 and Piezo2
ΔF
ΔF
- Expressedin numerous tissues 0
; intestine, brain, heart, lung, etc. 0 Line 2 inx-2 inx-20
0 20 0 20
Time (sec)
Time (sec)
0 20
- Activated by membrane stretch and pressure
n=20
Coste al., (2010), Science, Jing et al., (2014) Nature, n=20
Lee et al., (2014), PNAS Ranade et al., (2014) PNAS,
Time (sec)
Time (sec)
Time (sec)
Gudipaty et al., (2017) Nature, etc. 0 20 0 20 0 20
[ Protein similarity ] Blue light exposed miniSOG ablates valve cells
C. elegans Defects in food movement of inx-2 and inx-20 mutants were restored
isoform mPiezo1 hPiezo1 mPiezo2 hPiezo2 valv-1p::miniSOG;valv-1p::gfp
300 *** by cell specific expression of inx-2 and inx-20 cDNA, respectively
Short (i isoform) 43 46 39 34
Long (a isoform) 50 47 30 27 200 inx-20;ceh-24p
Intensity (mean) 15,000 Wild-type ::inx-20 cDNA
pezo-1 encodes 14 isoforms in C. elegans 100
Pharyngeal-intestinal valve Pharyngeal-intestinal valve n=20 ΔF
0 0
No Light inx-2;valv-1p inx-2;ceh-24p
pezo-1p(L) Light
::inx-2 cDNA
::inx-2 gDNA
Ablation of valve cells cause excess accumulation of GFP-
tm5111 tm5071 pezo-1p(S) microsphere similar to pezo-1 mutant animals
tm5750 n=20
Time (sec)
Time (sec)
tm10725 valv-1p:: 0 20 0 20
tm10726 lsk51 Wild-type miniSOG
2kb
15,000 No Light Light Light
inx-2 and inx-20 mutants show irregular defecation patterns
Results ΔF
n=20
0 Defecation cycle
0 20 0 20 0 20
Time (sec)
Time (sec)
Time (sec)
pBoC → Exp
Long isoforms of pezo-1 are expressed pBoC → pBoC
in pharyngeal-intestinal valve pezo-1 mutant animals have irregular defecation cycle 12 10 80 *** **
60
Wild-type tm10725 Interval (sec) 8 * * Interval (sec)
10 20 30 40 50 10 20 30 40 50 6 * ** *** 40
pm8
0 valve 60 60 4
………І………І………І………І………І………І ………І………І………І………І………І………І
0..E………………………………………….p.. 0...E…………………………………………….. 2 20
Pharyngeal-intestinal valve .E………………………………………….p…E. …p….E…………………………………………. lsk51 tm5111 tm5071 inx-2 tm5750 tm10725 lsk51 tm5111 tm5071 tm5750 inx-2 tm10725
…………………………………………..p…E… ………………………………….p….E………… Wild-type tm10726 inx-20 Wild-type tm10726 inx-20
p = pBoc …………………………………….p…E………. ……………………………………………………. n=20- 27
E = Expulsion ………………………………p………………….stop …………………………………………………….stop
How can we see food movement from the pharynx into intestine? Exp pBoC
Wild-type
Working model
Recording
for 20sec
(200ms/frame) tm10725
Adult
GFP-microsphere seeding plate 10mins feeding 2% agarose gel pad
FOOD
tm10725
pezo-1 mutant animals exhibit excess accumulation of rescued FOOD INX-20
Time (sec)
Time (sec)
GFP-microsphere in the anterior intestinal lumen 0 300 0 300 INX-2 PEZO-1
n=26
15,000 Wild-type tm10726 tm5071 lsk51 Gap junctions link all pharyngeal muscles
within a segment indirectly for pm2 to pm7
ΔF
0
Time (sec)
0 20 tm5111 tm5750 tm10725
Future plans
Time (sec)
0 20 0 20 0 20 Albertson and Thomson, (1976) Phil. Trans. Royal Soc. London
Time (sec)
Time (sec)
n=20 Altun et al., (2009), Dev. Dyn - Investigate relationship between PEZO-1 and Innexin
The valve cells are connectedto pharyngeal muscles - Examine the calcium activities of valve cells in pezo-1 mutants and innexin mutants
Expression pezo-1 long isoform rescued defects of pezo-1 mutant via INX-2 and INX-20 gap junction channels - Examine the PEZO-1 functions by ectopic expression pezo-1 in the olfactory sensory neurons
tm10725 inx-20p::gfp References
pezo-1p(L)::
valv-1p::
Wild-type - pezo-1 cDNA(L) pezo-1 cDNA(S) 1. Costeal., (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science.
15,000 2. Gudipaty et al., (2017) Mechanical stretch triggers rapid epithelial cell division through Piezo1. Nature.
Line 1 Line 1
3. Ranade et al., (2017) Piezo1 a mechanically activated ion channel is required for vascular development in mice. Nature.
pm8 : inx-2, inx-20,
ΔF ceh-24
K. Kim lab CGC
0 Line 2 Line 2 Valve : inx-2
Time (sec)
Time (sec)
0 20 0 20 Dr.Kyuhyung Kim NBRP
0 20
ceh-24p::gfp
Woochan Choi Jihye Cho
Line 3
Time (sec)
0 20 Do-Young Kim Jimin Kim
n=20 pm8 Woojung Heo Seo Yeong Kim
YongJin Cheon Semin Hwangbo
Kyeong Min Moon Seunghee Oh
Hyeonjeong Hwang Ok Hwa Kim
0 20
Time (sec)
Harfe and Fire, (1998) Genes Dev