Triggering Cation-Induced Contraction of Cytoskeleton Networks via Microfluidics

Abstract

The dynamic morphology and mechanics of the cytoskeleton is determined by interacting networks of semiflexible actin filaments and rigid microtubules. Active rearrangement of networks of actin and microtubules can not only be driven by motor proteins but by changes to ionic conditions. For example, high concentrations of multivalent ions can induce bundling and crosslinking of both filaments. Yet, how cytoskeleton networks respond in real-time to changing ion concentrations, and how actin-microtubule interactions impact network response to these changing conditions remains unknown. Here, we use microfluidic perfusion chambers and two-color confocal fluorescence microscopy to show that increasing magnesium ions trigger contraction of both actin and actin-microtubule networks. Specifically, we use microfluidics to vary the Mg²⁺ concentration between 2 and 20 mM while simultaneously visualizing the triggered changes to the overall network size. We find that as Mg²⁺ concentration increases both actin and actin-microtubule networks undergo bulk contraction, which we measure as the shrinking width of each network. However, surprisingly, lowering the Mg²⁺concentration back to 2 mM does not stop or reverse the contraction but rather causes both networks to contract further. Further, actin networks begin to contract at lower Mg²⁺ concentrations and shorter times than actin-microtubule networks. In fact, actin-microtubule networks only undergo substantial contraction once the Mg²⁺ concentration begins to lower from 20 mM back to 2 mM. Our intriguing findings shed new light on how varying environmental conditions can dynamically tune the morphology of cytoskeleton networks and trigger active contraction without the use of motor proteins.

Keywords: actin; cytoskeleton; microfluidics; microscopy; microtubules.

FIGURE 1 |

Experimental approach to triggering cation-induced contraction of cytoskeleton networks. (A) Cartoon of microfluidic device comprised of three channels separated by two semipermeable membranes (gray). The device has a central chamber containing the sample and two side channels used for buffer exchange. Buffer is pulled into the inlet and out of the outlet through capillary tubing using a syringe pump. The flow rate is set to allow for passive diffusion of buffer into the sample chamber as it flows from inlet to outlet. (B) Two-color laser scanning confocal images of an actin network (left) and an equimolar actin-microtubule network (right) where actin is magenta and microtubules are cyan. Scale bar is 50 μm.

FIGURE 2 |

Two-color confocal imaging of actin network undergoing contraction triggered by variation in Mg²⁺ concentration. Two-color laser scanning confocal images of an actin network (red) as the Mg²⁺ concentration slowly varies from 2 mM (green) to 20 mM (black) and back to 2 mM (green). Fluorescein in the 2 mM Mg²⁺ buffer (but not in the 20 mM Mg²⁺ buffer) is used to quantify the Mg²⁺ concentration as a function of time. Because it takes ~5 min for the buffer from the reservoir to enter the sample channel, the buffer channels in the first few images are black despite being at 2 mM Mg²⁺. At t = 30 min, the 20 mM Mg²⁺ solution is introduced, which is seen as the green signal intensity decaying to black. At t = 120 min, the 2 mM Mg²⁺ solution is reintroduced, viewed as increasing intensity in the green channel. Scale bar is 500 μm and time is in minutes.

FIGURE 3 |

Actin and actin-microtubule networks contract in response to continuous variation of Mg²⁺ concentration. The average width (left axis) of the actin network samples (magenta) and actin-microtubule network samples (cyan) as a function of time. The Mg²⁺ concentration (right axis) is also plotted (green line) as a function of time. The three phases of the experiment (I-III) are separated by dashed lines: I) 2 mM Mg²⁺ solution (original polymerization buffer) diffuses through the sample for 30 min II) exchange to 20 mM Mg²⁺ buffer is initiated and proceeds until 120 min III) 2 mM Mg²⁺ buffer is reintroduced for 50 min.

FIGURE 4 |

Cytoskeleton networks exhibit network-dependent contraction in response to both increasing and decreasing Mg²⁺ concentration (A) The average width of actin network samples (magenta) and actin-microtubule network samples (cyan) as a function of Mg²⁺ concentration. Experimental time is indicated in the symbol gradient coloring where t = 0 is cyan and magenta and t = 170 is light cyan and magenta. (B) The fractional amount of contraction measured for each Phase (I, II, III; described in the text), computed by W_f/W_i where W_i is the width at the beginning of the experiment and W_f is the width at the end of the corresponding Phase. The dashed lines separating the three Phases approximate the relative length of time of each phase. The corresponding Mg²⁺ concentration is depicted as a gradient with dark to light green indicating 2 to 20 mM Mg²⁺.