Fig 1.
Microfluidic device for studying cell blebbing in confinement.
(A) Confocal micrograph of the microfluidic gradient generator. The device consists of an array of microchannel gradient channels aligned perpendicular to main flow channels. Buffer is introduced through “Buffer inlet” (shown in green), and buffer with cAMP is introduced through “cAMP inlet” (shown in purple). Cells are loaded into the device by flowing a cell suspension into the “Cell inlet”. (B) Linear concentration profiles in gradient channels are established through molecular diffusion between the two main flow channels. Each gradient channel has a length of 150 μm and a width of 50 μm. (C) The “thin” confinement channels are between 1.7 and 3.8 μm in height connected to the main “thick” flow channels that are 80 μm in height.
Fig 2.
Cell migration in microfluidic confinement.
(A) A confined Dictyostelium cell migrates up a cAMP gradient. The gradient is imaged using Alexa Fluor 647 hydrazide. The seven superimposed micrographs of the cell were captured (3.28 FPS) at time intervals t = 0, 1:54, 2:25, 3:05, 4:27, 12:10 and 17:16 min. (B) During bleb expansion F-actin scars remain behind and the newly formed bleb is almost devoid of F-actin. (C) When confined, blebs formed at the leading edge of a cell expressing a membrane marker (mCherry-cAR1) and F-actin reporter (GFP-LimEΔcoil). (D) Cells predominately formed pseudopods when migrating under buffer.
Fig 3.
Blebs produced by a cell moving through a 1.7 μm microchannel.
(A) A bleb forms when the cell membrane detaches from the cortex. (B) The bleb expands in approximately 0.2 s, leaving behind a cortical F-actin scar. (C, D) Initially the bleb lacks an F-actin cortex, but it is rebuilt in less than one second. Images captured at 5.12 FPS.
Fig 4.
Cell confinement is controlled through microchannel height, as determined from confocal images.
(A) Top view of cells confined using three different microchannel heights. (B) Side view of cells confined to heights of 1.7, 2.4 and 3.8 μm, as determined by using confocal microscopy. Fluorescence signal is from GFP-LimEΔcoil. (C) The measured cell height correlates with the fabricated microchannel height. Cell numbers shown on each bar. Error bars represent SEM.
Fig 5.
Cell migration is influenced by microfluidic confinement.
Dictyostelium cells are observed as they migrate up a 20 nM/μm cAMP gradient. (A) Chemotactic cell velocity decreases as the height of the channel is reduced. (B) Blebbing increases as the height of the microfluidic gradient channel is reduced. Blebs given as percentage of total projections (pseudopods + blebs). Error bars represent SEM. The number of cells quantified shown on bars. **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.
Fig 6.
Bleb-driven movement is regulated by cAMP gradient steepness.
The steepness of the cAMP gradient increases the blebbing frequency. Cell numbers are shown on bars. Error bars represent SEM. ***P ≤ 0.001, ****P ≤ 0.0001.
Fig 7.
Cell speed is reduced when cells migrate largely using blebs.
The Dictyostelium cell velocity is reduced with increasing the steepness of cAMP gradient at 1.7 μm and 2.4 μm. Under these conditions cells migrate using a larger percentage of blebs (see Fig 6). Cell velocity remains approximately constant when cells migrate in 3.8 μm-tall channels, were cells used very few blebs. The data shown are the mean ± SEM.
Fig 8.
Chemotaxis index (C.I) of Dictyostelium is not influenced by microchannel height.
The cell tracks for 20 cells are shown for cells chemotaxing in a 1.7 μm-tall channel in (A) no cAMP gradient and (B) a 20 nM/μm cAMP gradient. (C) The cell chemotaxis index varies from 0.9–1.0 when cells migrate in a cAMP gradient, but significantly less when no gradient is applied. The data shown are the mean ± SEM.
Fig 9.
Myosin-II localizes to the cell cortex when cells are confined within microfluidic gradient channels.
(A) Fluorescence intensity of GFP-myosin was higher at the cell cortex (Icort) than in the cytosol (Icyto). (B) The ratio of Icort/Icyto increased when the microfluidic confinement channel height was decreased from 3.8 μm to 1.7 μm. Cell numbers are shown on bars. Error bars represent SEM. ****P ≤ 0.0001.
Fig 10.
Myosin-II and cytosolic mCherry during migration under microfluidic confinement.
(A) Myosin-II accumulates at the cortex in confined Dictyostelium cells, while (B) mCherry, used as a volume marker, does not show cortical/membrane enrichment at 1.7 μm. (C) Myosin-II and (D) mCherry do not concentrate at the cell cortex in confined cells at 3.8 μm. Cell numbers are shown on bars. Error bars represent SEM.
Fig 11.
Increasing the cAMP gradient from 0.7 nM/μm to 20 nM/μm induces the cells to produce blebs with larger surface area.
Sorbitol and blebbistatin reduced the bleb surface area. Bleb numbers are shown on bars. Error bars represent SEM. ****P ≤ 0.0001.
Fig 12.
Effects of high osmolarity and blebbistatin on the chemotactic velocity of cells and on the percentage of blebs utilized by the cells.
(A) High osmolarity buffer led to an increase in cell velocity at 1.7 μm and did not impact the velocity of the cells at 3.8 μm. Additionally, blebbistatin did not impact chemotactic velocity at 1.7 μm and reduced cell velocity at 3.8 μm. (B) Cells utilized higher percentage of pseudopods in high osmolarity buffer and after treatment with blebbistatin in 1.7 μm-tall channels. Cell numbers are shown on bars. Error bars represent SEM. *P ≤ 0.1, ***P ≤ 0.001, ****P ≤ 0.0001.