Principios básicos para su organización

To determine whether cells detect a difference between constant and pulsatile me- chanical forces, we designed an experiment where the force magnitude was held constant (120 pN) and the oscillatory nature of the force was varied between a (1) 100% duty cycle, 0 mHz constant force, and a (2) 16.7% duty cycle, 83.3 mHz pulsatile force. Functionally, this was achieved with a 60◦ arc magnet at a height of 10 mm either in a stationary position (constant force) or rotating at 5 rpm (pulsatile force) (Fig3.6A,B). In order to prevent cells in the specimen plate that were not exposed to force (roughly, those outside the 60◦ arc directly underneath the magnet) from diluting the protein lysate during the biochemical analyses, we created a partition using a rubber mold. Briefly, we removed a 60◦ section from a 2 mm-thick rubber mold made in an empty 10 cm culture dish, and then plated cells in this section at the same 70% confluence as the rotating condition (Fig 3.6 C). With this consideration, only the cells exposed to the constant force went into constant force cell lysate. During protein-loading in the western blot assay, the protein amount added in the pulsatile force condition lanes were normalized to the amount added in the constant force lanes.

Figure 3.6: Constant and pulsatile magnet assay configurations. To detect a possible difference between constant and pulsatile mechanical forces, we held the force magnitude constant at 120 pN (10 mm magnet-to-cell distance). (A) The constant force configuration (100% duty cycle, 0 mHz frequency) is achieve by setting the motor speed to 0 rpm. (B) The pulsatile force configuration (16.7% duty cycle, 83.3 mHz frequency) is achieved by setting the motor speed to 5 rpm. (C) To ensure only the cells exposed to the constant force went into constant force cell lysate, a 60◦ partition was made using a rubber mold.

3.7 Pulsatile Mechanical Force Enhances ERK and RhoA Activation

In order to assess the biological relevance of this device, we chose to test the sys- tem using ECs. ECs are exposed to continuous mechanical stimulation as a result of blood flow, and the nature of the mechanical force can differ (oscillatory vs. laminar) depending on location within the vasculature. Although parallel- and cone-and-plate flow systems have been used to apply laminar or oscillatory shear stress of known magnitudes to ECs, our device offers receptor-pathway specificity by varying the func- tionalization of the externally attached magnetic beads. Work from our lab and others have indicated that the EC adhesion molecule PECAM-1 plays an essential role in EC mechanotransduction of blood flow (Tzima et al., 2005)(Harry et al., 2008)(Stevens et al., 2008)(Goel et al., 2008). Thus, PECAM-1 is an ideal candidate to study the biological relevance of varying force dynamics.

Mechanical forces trigger the activation of numerous signaling pathways, many of which converge to ultimately regulate the activity of the master regulators of cytoskele- tal dynamics, such as the GTPase RhoA (Lessey et al., 2012). Our previous work has shown that force application on PECAM-1 influences effective EC stiffness and that the stiffening response requires the activation of numerous signaling molecules, includ- ing ERK and RhoA. Using the magnetic tweezers to apply force on PECAM-1, ECs were observed to significantly increase their effective stiffness within 1 min of force application (Collins et al., 2012), and this stiffening effect required both ERK and RhoA activity. However, when biochemical analyses were performed to investigate

force-dependent activation of these signaling molecules, application of constant force using a permanent magnet did not induce significant activation of ERK or RhoA until later time points (5 or 30 min of force application) (Collins et al., 2012). These data suggested that nature of force application on PECAM-1 (pulsatile vs. constant) might influence the dynamics of downstream signaling. Our device allowed us to directly test this hypothesis by selectively manipulating the duty cycle and frequency of the force, while keeping the force magnitude as well as other experimental variables constant. Here, using a 60◦ arc magnet that is rotated at 5 rpm (Fig. 3.6 B) and generates a 100 pN force at 10 mm to give a force regimen consistent with the magnetic tweezers in (Collins et al., 2012), we tested the effect of pulsatile force application on dynamics of ERK and RhoA activation.

Figure 3.7: Pulsatile mechanical force enhances Erk activation. Adherent ECs on FN were incubated with anti-PECAM-1-coated magnetic beads and subjected to a constant or pulsatile force for either 1 or 2 minutes. (A) Cells were lysed, subjected to SDS-page, and immunoblotted with pERK or ERK antibodies (n = 3). (B) Fold increase in activated ERK1/2 was quantified using NIH ImageJ software. Values were normalized to the No Force condition. Bar graphs display averages from at least three independent experiments and error bars indicate s.e.m, *p < 0.05. Data: Caitlin Collins

Figure 3.8: Pulsatile mechanical force enhances RhoA activation. Adherent ECs on FN were incubated with anti-PECAM-1-coated magnetic beads and subjected to a constant or pulsatile force for either 1 or 2 minutes. (A) Active RhoA (RhoA-GTP) was isolated with GST-RBD and analyzed by western blot (n = 3). Whole cell lysates were subjected to SDS-PAGE to detect Total RhoA protein levels. (B) Fold increase in RhoA activity was quantified using NIH ImageJ software. Values were normalized to the “No Force” condition. Bar graphs display averages from at least three independent experiments and error bars indicate s.e.m,∗p < 0.05. Data: Caitlin Collins

ECs plated were plated on fibronectin (FN)-coated 10cm dishes and incubated with anti-PECAM-1-coated beads for 30 min. Cells were then subjected to constant or pulsatile force using our rotated magnet device. Cells were lysed and processed for immunoblot analysis to assay ERK activation. We did not detect significant activation of ERK activation at 1 or 2 min of force application when the position of the arc magnet was held in a fixed in position to generate a constant force (Fig. 3.7). However, when magnet was rotated in order to produce pulses of forces, ERK was significantly activated after 2 min of force application when compared to the “No force” control (Fig. 3.7).

The small GTPase RhoA is activated in response to force (Collins et al., 2012)(Guil- luy et al., 2011)(Osborne et al., 2014) and RhoA activity is required for the cellular

response to tension on PECAM-1 (Collins et al., 2012). In order to test the hypothesis that dynamic force application accelerates activation of RhoA, cells were incubated with anti-PECAM-1-coated beads and subjected to constant (fixed magnet) or pulses (rotating magnet) of force. Rho pulldowns were performed to assess the population of activated, GTP-bound RhoA and lysates were subjected to western blot analysis. Con- sistent with our previous work, we were unable to detect significant RhoA activation when force was applied to PECAM-1 for 1 or 2 min (Fig. 3.8). In contrast, RhoA activ- ity quickly increased by 3-fold in as little as 1 minute of pulsatile force application, when compared to the “No force” control (Fig. 3.8). Force-dependent ERK activation has been shown to influence RhoA activity by activating the guanine nucleotide exchange factor GEF-H1 (Collins et al., 2012)(Guilluy et al., 2011). Here, we see significant RhoA activation at earlier time points than ERK activation in response to pulsatile force. However, this difference in the timescale of significant Erk phosphorylation (2 minute) and RhoA activation (1 minute) may be due to the fact that other GEFs are known to active RhoA in response to force, such as LARG (Guilluy et al., 2011). These results strongly suggest other proteins in the RhoA pathway are sensitive to pulsatile mechanical forces and may be contributing to early activation of the GTPase.