Substrate-bound protein gradients to study haptotaxis

Abstract

Cells navigate in response to inhomogeneous distributions of extracellular guidance cues. The cellular and molecular mechanisms underlying migration in response to gradients of chemical cues have been investigated for over a century. Following the introduction of micropipettes and more recently microfluidics for gradient generation, much attention and effort was devoted to study cellular chemotaxis, which is defined as guidance by gradients of chemical cues in solution. Haptotaxis, directional migration in response to gradients of substrate-bound cues, has received comparatively less attention; however, it is increasingly clear that in vivo many physiologically relevant guidance proteins - including many secreted cues - are bound to cellular surfaces or incorporated into extracellular matrix and likely function via a haptotactic mechanism. Here, we review the history of haptotaxis. We examine the importance of the reference surface, the surface in contact with the cell that is not covered by the cue, which forms a gradient opposing the gradient of the protein cue and must be considered in experimental designs and interpretation of results. We review and compare microfluidics, contact printing, light patterning, and 3D fabrication to pattern substrate-bound protein gradients in vitro. The range of methods to create substrate-bound gradients discussed herein makes possible systematic analyses of haptotactic mechanisms. Furthermore, understanding the fundamental mechanisms underlying cell motility will inform bioengineering approaches to program cell navigation and recover lost function.

Keywords: digital gradient; haptotaxis; immobilized gradient; reference surface; substrate-bound gradient.

Figure 1

A surface gradient of a cue (yellow) entails an opposite gradient of the underlying reference surface (RS) (blue). The RS, acting relative to the guidance cue, either promotes or inhibits cell adhesion – in either case the RS gradient is expected to modulate the cell response. Consequently, haptotaxis of a cell on a surface-bound gradient is a response to both the gradient of the guidance cue of interest and the opposing RS gradient.

Figure 2

Schematic representations and experimental results of porous membrane doping and microfluidic methods to form substrate-bound protein gradients. (A) By applying a vacuum, protein solutions can be aspirated through a silicon matrix where the proteins bind. By placing an angled coverslip on top of the solution, a protein gradient can be adsorbed to the matrix. Reprinted with permission from AAAS (Baier and Bonhoeffer, 1992). (B) Micrograph image of a microfluidic serial dilutor forming a dilution series that is flowed into a wide chamber forming a gradient across it. With the appropriate surface chemistry, proteins adsorb to the surface, thereby capturing the diffusible gradient on the substrate. (C) A microfluidic probe with injection and aspiration apertures serves to create confined microfluidic flows that can pattern gradients (here fluorescence intensity is shown as topography) by continuously changing the writing speed while patterning. Scale bars are (A,B) 100 μm and (C) 500 μm.

Figure 3

Schematic representations and experimental results of hydrogel-based diffusion, microcontact printing, and dip-pen nanolithography methods to form substrate-bound protein gradients. (A) Hydrogel stamps in contact with the substrate create closed channels that can be filled with a protein solution. The proteins then diffuse through the hydrogel and adsorb to the surface in a graded distribution. Republished with permission of the Journal of Neuroscience (Mai et al., 2009); permission conveyed through Copyright Clearance Center, Inc. (B) In microcontact printing, polymer stamps with topography are inked with a protein solution and used to stamp protein in a gradient geometry by limiting the regions of contact between the stamp and the surface. (C) Dip-pen nanolithography employs an AFM cantilever inked with a protein solution to transfer localized nanovolumes of protein solution onto the surface in any desired geometry (e.g., dot gradient). Reprinted with permission from AAAS (Huo et al., 2008). Scale bars are (A) 100 μm, (B) 1 mm, and (C) 30 μm.

Figure 4

Haptotaxis gradients can either be continuous or digital. (A) Continuous gradients have constant changes in protein concentration, and usually result from the adsorption of biomolecules from diffusible gradients. Digital gradients can be formed in a number of ways, (B) by patterning biomolecules as lines or dots and (C) changing their size, (D) spacing in 1D, or (E) both. (F) The spacing can also be changed in 2D.

Figure 5

Non-monotonic haptotaxis gradients. Non-monotonic protein gradients achieved with (A) low-cost lift-off nanocontact printing showing (top) the dots of the digital design (black), and (bottom) the fluorescence image of a red fluorescently labeled IgG of the same non-monotonic DNG. (B) Non-monotonic gradients can also be achieved through laser-assisted protein patterning (LAPAP), here illustrated by patterning a fluorescently labeled IgG. The red curves illustrate the density functions of the patterned gradients. Scale bars are (A,B) 50 μm.

Figure 6

Schematic representations and experimental results of porous membrane doping and microfluidic methods to form substrate-bound protein gradients. (A) By applying a heat gradient under the surface, microspheres melt and interact with the surface to different extents thereby creating a gradient of dots of different size. Adapted with permission from Taylor et al. (2012). Copyright 2012 American Chemical Society. (B) Controlled photobleaching of fluorescently labeled proteins can also be used to increase the reactivity of the proteins with the surface in specific locations and by controlling light intensity or exposure, gradients can be formed. Reproduced in part from Bélisle et al. (2008) with permission of The Royal Society of Chemistry. (C) The presence of a microfluidic channel within a hydrogel enables the filling of a point source solution within a 3D environment. The proteins diffuse from the source and create a gradient in a similar fashion to hydrogel stamp diffusion. Reproduced in part from Lienemann et al. (2015) with permission of The Royal Society of Chemistry. Scale bars are (A) 20 μm, (B) 25 μm, and (C) 250 μm.