Page 3 - E. Cell adhesion and cytoskeletal dynamics

Cytoskeleton Dependent Activation of Tentonin 3/TMEM150C,

an Essential Component of Mechanosensitive Channel

Sungmin Pak , Gyu-Sang Hong , Hyungsup Kim , Jaehyouk Choi , Huanjun Lu , Kyungmin Kim , Hyesu Kim ,
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Luan Nguyen Thien , Taewoong Ha , Sujin Lim , Mi-Sun Kim , and Uhtaek Oh 1
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1 Sensory Research Center, CRI, Brain Science Institute, Korea Institute of Science and Technology, Seoul 02792, Korea
Abstract Introduction-1
Tentonin3/TMEM150C (TTN3) is a mechanosensitive ion channel that has characteristics of slowly
inactivating kinetics by mechanical stimulation. Recent studies have proposed whether TTN3 is an ion DRG Sensory neurons Mechanosensitive channel
channel and its independent channel activity is validated. The opening mechanism of mechanosensitive
ion channels have been suggested into two theories. One theory is regarding the activation through ?
direct cell plasma membrane stretch, for example, the Piezo family. Another theory pertains the influence
of tethering proteins near the plasma membrane activating mechanosensitive channels, for example,
MEC complex in C. elegans. Focal Adhesion (FA) Complex is a multi-protein assembly of
transmembrane proteins and intracellular proteins that provides connection between the cell and the
extracellular matrix. We report that the FA Complex of Piezo1-Knockout (P1KO) HEK293T cells is
hampered and selective knockdown of FA related proteins, Talin1, Vinculin and Integrin, diminished
TTN3 mechanical current in Ttn3-transfected HEK293T cells. Therefore, we propose the disturbed FA Piezo2
Complex in P1KO HEK293T cells is responsible for the absence of TTN3 mechanical current in ttn3- (Delmas, 2011, Nature Review)
transfected P1KO-HEK293T cells.
There are mainly three types of MS currents in isolated DRG neurons, determined by inactivation time
Introduction-2 kinetics under whole-cell patch mode: rapidly adapting (RA) currents (~10 ms), intermediately adapting (IA)
currents
(10 ms–30 ms) and slowly adapting (SA) currents (>30 ms) (Huand Lewin, 2006). Although Piezo2 has been
TTN3 is a novel mechanosensitive (MS) ion discovered as a RA MS channel (Coste, 2010), molecular identity of SA MS channel is not known yet.
Introduction-3
channel Candidate
Is TTN3 a Modulator of MS channels?
No activation of TTN3 in HEK-P1KO Kinetics of TREK1 & PiezosTTN3
modulates inactivation
(Dubin et al., Neuron, 2017)
(Hong et al., Neuron, 2016) Independent of TTN3 from Piezo1
1. Activated by mechanical stimuli with slow-inactivation kinetics.
2. Required for SA MS currents in DRG neurons.
3. Present in muscle spindles and contributes to proprioception.
1
A B
HEK HEK- (Hong et al., Neuron, (Anderson et al., Cell reports, 2018)
△P1 3 2017)
Z- 4 3 4 3 A B C D Figure 3. Focal Adhesion (FA)
Stacked 3 6 3 6 TTN3/S 600 TTN3/VCLsi 600 TTN3/TL1si dependency of TTN3 MA current
F-actin
ddCt (to GAPDH) H 500 pA 100 pA 100 pA Vinculin siRNA or Ttn3 + Talin-1 siRNA (C)
C 600 ms crbl ms ms ** *** (A–C) Representative traces of whole-cell MA
currents in Ttn3 + scrambled siRNA, ttn3 +
HEK-
transfected HEK293T cells.
E
(D-F) Summary of average peak amplitude (D),
△P1
K
(G) Peak current amplitudes of Ttn3 co-
on
transfected with Vcl, Tl1, and Kk1.
Figure 1. Defects of intracellular cytoskeletal structure in HEK-P1KO cells. E *** ** *** F G Co-expressi threshold (E) and peak distance (F).
(A) Combined Z-stack images of HEK293T and P1KO-HEK293T cells. ANOVA one-way ANOVA with Dunnett’s
(B) Summary of number and intensity of foci counted between HEK293T and P1KO-HEK293T cells. correction, **p < 0.005, ***p < 0.001). Data
(C) mRNA expression levels of hPiezo1 (hP1), TTN3, Vinculin (VCL), Talin1 (TL1), and Kank1 (KK1) in shown as mean ± SEM.
HEK293T and P1KO-HEK293T cells.
2 4 5
A
Tal F-actin M Figure 5. Generation
in- e of TTN3 MS current in
1 r
g HEK-P1KO cells.
H 10 e μm HEK- (A) Immunofluorescent
B E △P1 images, stained with
Talin- K Vincu M Merg
Talin
Vinc
1 lin e Phalloidin(F-actin) in
ulin er HEK-P1KO cells after 2
g hours treatment of DMSO
H e HEK-
(i) E (ii) △P1 or 250nM Jasplakinolide.
K (B–D) Representative
traces of whole-cell MS
currents in response to
2 μm 2 μm mechanical indentation
Figure 2. Difference in cytoskeletal structure steps from 6 to 8 μm in
between HEK293T and P1KO-HEK293T cells. TTN3 (B and D) or GFP
(A) and (B) Immunofluorescence images of cells stained (C) overexpressing HEK-
with Talin1 antibody and Phalloidin (A) and Talin1 and P1KO cells. 250 nM
Vinculin antibody (B) in HEK293T and P1KO-HEK293T Figure 4. TTN3 dependent MS currents in HEK cells Jasplakinolide (JAS) or
cells. DMSO was applied to the
Conclusion (A) Representative traces of maximal MS currents in mTTN3 cultured medium for 2
expressing or non-expressing HEK cells. Blue arrowhead represents hours before
the mechanical indentation starting point of 600 ms duration with 5 μm electrophysiology test.
distance. E hold = –60 mV.
1. TTN3 mediates slowly inactivating MS current in (B) Displacement dependent MS currents of TTN3 and naïve HEK cell (E and F) Quantification of
the absence of Piezo1 by F-actin polymerization (C) Quantification of maximal MS currents measured at 5 μm saturated peak amplitude
mechanical stimulation. (***p < 0.001, Student’s t test). Red arrows (E) and threshold (F).
2. TTN3 channel activity is dependent on focal indicate each representative traces shown in figure 1E. (***p < 0.001, Student’s t
adhesion complex (D) RT-qPCR analysis by indicated primers, hPIEZO1 or mTtn3 in HEK test).
and HEK-P1KO cells.
3. TTN3 acts as an essential component of the slowly (E and G) Representative traces of hPiezo1 and mTTN3 MS current
inactivating MS channel complex. induced by mechanical stimulation before and after treatment of
Yoda1.
(F and H) Quantification of Piezo1 and TTN3 MS current ratio (I and L)
and Tau i (J and M) before and after treating Yoda1. (**p < 0.005, ***p
< 0.001, Student’s t test).