Molecular gradients are important for various biological processes including the polarization of tissues and cells during embryogenesis and chemotaxis. Investigations of these phenomena require control over the chemical microenvironment of cells. We present a technique to set up molecular concentration patterns that are chemically, spatially and temporally flexible. Our strategy uses optically manipulated microsources, which steadily release molecules. Our technique enables the control of molecular concentrations over length scales down to about 1 μm and timescales from fractions of a second to an hour. We demonstrate this technique by manipulating the motility of single human neutrophils. We induced directed cell polarization and migration with microsources loaded with the chemoattractant formyl-methionine-leucine-phenylalanine. Furthermore, we triggered highly localized retraction of lamellipodia and redirection of polarization and migration with microsources releasing cytochalasin D, an inhibitor of actin polymerization.
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We thank S. Dandekar, A. Houk, A. Millius and S. Wilson for help with cell culturing and sample preparation, A. Schaefer for helpful discussions, and M. Elimelech and W. Mitch for use of equipment. This work was supported by a fellowship (BMBF-LPD 9901/8-162) from the German Academy of Sciences Leopoldina to H.K., a US National Institutes of Health grant (1U54-RR0222332) and US National Science Foundation grants (CBET-0619674 and DBI–0619674) to E.R.D., a US National Institutes of Health grant (RO1-GM084040) to O.D.W and a US National Science Foundation CAREER grant (CBET-0747577) to T.M.F.
Supplementary Figures 1–10 and Supplementary Notes 1–7 (PDF 8345 kb)
A single optically manipulated bead which is loaded with chemoattractant is able to induce directed polarization and migration of a neutrophil. An individual PLGA particle loaded with fMLP is optically micromanipulated and moved close to the membrane of a HL-60 cell. The cell starts to polarize and migrate in the direction of the particle. The particle is moved in a counter clockwise way around the cell and the cell is changing its polarization and migration direction in response to the altered particle position. Scale bar, 10 μm. (MOV 355 kb)
A chemoattractant-loaded particle induces cell motility and change of the direction of migration of a neutrophil. An individual PLGA particle loaded with fMLP is optically micromanipulated and moved close to the membrane of a HL-60 cell. The cell starts to polarize and migrate at an angle of about 45° relative to the gradient direction as defined by the bead position. After about 200 seconds, the cell changes its direction of motion and migrates in the direction of the particle. During this motion, the cell forms a stable lamellipodium at its leading edge. Scale bar, 10 μm. (MOV 883 kb)
Bipolar stimulation with chemoattractant induces formation and broadening of a lamellipodium. Upon positioning of the fMLP-loaded beads close to a HL-60 cell, the cell forms a lamellipodium in the direction of the beads. As the cell advances, it broadens its lamellipodium approaching both beads. Ultimately, the lamellipodium reaches its maximal size, and the cell orients towards the lower left bead. Scale bar, 10 μm. (MOV 940 kb)
A control particle does not stimulate a cell response. An individual optically manipulated plain PLGA particle (no fMLP encapsulated) is used to try to stimulate a HL-60 cell. The cell does protrude and retract in varying directions, but it does not protrude or migrate in the direction of the particle. Scale bar, 10 μm. (MOV 574 kb)
A control particle does not stimulate a cell response. An individual optically manipulated plain PLGA particle (no fMLP encapsulated) is used to try to stimulate a HL-60 cell. The cell does protrude and retract in varying directions, but it does not protrude or migrate in the direction of the particle. Scale bar, 10 μm. (MOV 614 kb)
Optically manipulated microsources of cytochalasin D induce highly localized retraction in the center of a lamellipodium. Two beads releasing the actin polymerization inhibitor cytochalasin D are positioned close to the center of the lamellipodium of a HL-60 cell. The lamellipodium starts to retract in a small region around the beads yielding a lamellipodium that is split into two parts. Finally, one of the two lamellipodia retracts and only one remains. Scale bar, 10 μm. (MOV 1605 kb)
Migrating cell squeezes between two optically manipulated microsources of cytochalasin D. The two beads are placed in front of a migrating HL-60 cell. Then, the bead-to-bead distance is increased to about 20 μm. As the cell migrates towards the two particles, the lamellipodium starts to retract on the two sides closest the beads while the central part of the leading edge continues to extend. Finally, the cell rounds up and retracts its leading edge. Scale bar, 10 μm. (MOV 1826 kb)
Cell response to time-varying bipolar stimulus of cytochalasin D. A HL-60 cell stops its migration and retracts its lamellipodium in response to two cytochalasin-releasing beads which are placed in the direction of cell migration. Then, the two beads are positioned on two opposing ends of the cell in the direction of cell polarization. Subsequently, the cell re-polarizes in a direction perpendicular to the axis defined by the beads. Scale bar, 10 μm. (MOV 945 kb)
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Kress, H., Park, J., Mejean, C. et al. Cell stimulation with optically manipulated microsources. Nat Methods 6, 905–909 (2009). https://doi.org/10.1038/nmeth.1400
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