Microcontact printing of axon guidance molecules for generation of graded patterns

Philipsborn, Anne C. von; Lang, Susanne; Bernard, André; Loeschinger, Jürgen; Dávid, Christian; Lehnert, Dirk; Bastmeyer, Martin; Bonhoeffer, Friedrich

doi:10.1038/nprot.2006.251

Cited by 134 publications

(130 citation statements)

References 23 publications

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“…Here we developed a novel micro-contact printing strategy 30 to study local effects of the laminin-332 containing matrix. We showed that RA-mechanosensitive currents in the same cell are only suppressed in neurites on a laminin-332 stripe, but not on the control laminin stripe.…”

Section: Discussionmentioning

confidence: 99%

“…To further examine the mechanism of action of laminin-332 we applied the technique of micro-contact printing of substrate proteins [30][31] . We generated cross-hatched grid patterns with laminin stripes in one direction crossed with stripes of either laminin alone or laminin mixed with laminin-332 at 90 o to the laminin stripe (laminin-332 henceforth always refers to a mix of laminin: 20 μg/ml; laminin-332: 1.33 μg/ml; Molar ratio 15:1) (Fig.…”

Section: Laminin-332 Acts Locally Not Globallymentioning

confidence: 99%

“…Ganglia were washed once with PBS before incubation with 1µg/ml collagenase type IV in 1ml PBS at 37°C for 30 min. Ganglia were centrifuged briefly (170 x g), the supernatant was Microcontact printing can be pattern surfaces with proteins 30 . Negative silicon masters were provided by Dr. Siegmund Schroeter (Institute of Photonic Technology, Jena, Germany) from which PDMS stamps were cast, as described 30 .…”

Section: Cell Culturementioning

confidence: 99%

“…Ganglia were centrifuged briefly (170 x g), the supernatant was Microcontact printing can be pattern surfaces with proteins 30 . Negative silicon masters were provided by Dr. Siegmund Schroeter (Institute of Photonic Technology, Jena, Germany) from which PDMS stamps were cast, as described 30 . To print laminin protein on coverslips, these stamps were covered with a printing ink containing either 20 μg/ml EHS laminin, 20 μg/ml EHS laminin plus 1.33 μg/ml laminin-332 or 20 μg/ml laminin plus 0.67 μg/ml laminin-332 in PBS.…”

Section: Cell Culturementioning

confidence: 99%

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Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

et al. 2011

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2 These authors made an equal contribution. Basement membrane molecules such as laminin are important structural components of the skin 1-4 , but also serve as substrates for sensory neurons of the dorsal root ganglia (DRG) to grow in culture 5 . The main function of sensory neurons innervating the skin is to detect and relay relevant sensory stimuli, in particular mechanical stimuli 6 . It has long been known that sensory neurons with a nociceptive function (detecting potentially harmful stimuli) can have their endings in the epidermis 7-9 whereas mechanoreceptor endings (touch receptors) reside exclusively in the dermal layer [9][10] . Interestingly, the matrix environments of the epidermis and the dermis are very distinctive 11 . We showed that mechanosensitive currents required for touch receptor function depend on the presence of a protein tether which may function to couple mechanosensitive channels to a laminin-containing matrix 12 . The tether protein is not required for the mechanosensitivity of most nociceptive sensory neurons. Here we set out to address the idea that sensory mechanotransduction might be modulated by distinct matrix components made by different types of skin cells in different skin layers. We show that epidermal keratinocytes produce a matrix that is non-permissive for mechanotransduction and identify the factor responsible as laminin-332 (formerly known as laminin-5). Laminin matrices doped with small amounts of laminin-332 have a dramatically altered network structure that is non-permissive for tether attachment.We demonstrate a spatially restricted loss of mechanotransduction in neurite segments connected to laminin-332-containing matrices. Mutations in all three genes coding the trimeric laminin-332 protein complex can cause epidermolysis bullosa, a severe inherited skin blistering disease 1,3 . Human keratinocytes that produce a laminin-332 free matrix have no inhibitory activity on mechanotransduction. We have also discovered an activity of laminin-332 matrix in inhibiting sensory axon bifurcation. Our results reveal novel mechanisms whereby permissive and non-permissive substrates can spatially coordinate mechanotransduction in distinct domains within a single neuron. Results Keratinocyte matrix is suppresses mechanotransductionUsing whole-cell, patch-clamp techniques we directly recorded mechanosensitive currents in cultured sensory neurons [12][13][14][15][16][17][18][19][20] . We first asked whether co-culture of sensory neurons with different cellular components of the skin can modulate the activity of mechanosensitive currents. When sensory neurons are cultured on a laminin substrate, standardly-derived from Engelbreth-Holm-Swarm cells (EHS matrix, henceforth referred to as laminin), more than 90% of the cells exhibit a mechanosensitive current evoked using a small (~740 nm displacement) stimulus to the neurite 12, 14-15 . At least three types of mechanosensitive current can be measured in sensory neurons, classified according to their inactivation time constant τ 1 , rap...

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Section: Discussionmentioning

confidence: 99%

Section: Laminin-332 Acts Locally Not Globallymentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

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Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

et al. 2011

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“…These zig-zag stripes are valuable when analyzing cell behavior and signal-transduction events at substrate borders, where, for example, one part of a cell is adhering to a laminin substrate and the other part is contacting a substrate of different molecular nature 30 . Also, a novel technique allowing for microcontact printing of proteins on surfaces has been reported and offers a versatile tool to study the behavior of, for example, navigating growth cones in gradients of axon guidance cues 31 .…”

Section: Introductionmentioning

confidence: 99%

Stripe assay to examine axonal guidance and cell migration

et al. 2007

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Stripe assays have been widely employed as in vitro test systems to study the responses of growing axons, as well as migrating cells, to established or novel guidance molecules. We provide detailed protocols for both the original and the modified version of this assay, as they allow the analysis of the 'guidance properties' of active components present in crude membrane fractions or as purified molecules. Silicon matrices are used to produce striped patterns of active molecules on a surface (referred to as 'carpet'), followed by culturing of neurons, or any other cell type, on these carpets. After 1-2 days in culture, striped outgrowth of extending neuritesindicative of guided migration of cell processes-can be observed. We also discuss potential other applications (e.g., in neuronal regeneration and development) and modifications of the assay. The preparation of 10-12 carpets takes approximately 4-5 h. INTRODUCTIONThe stripe assay was originally designed by F. Bonhoeffer and co-workers 1,2 in the late 1980s to analyze fundamental axonal guidance mechanisms governing the formation of the topographic map established in the chick retino-tectal system (for review, see ref.3). Employing this assay, the presence of a graded distribution of repulsive axon guidance cues, present predominantly in posterior tectal membranes, was discovered. Subsequently, this assay was modified, permitting the identification of ephrin-As as the key components present in posterior membranes that are responsible for the repulsive properties 4-7 .The stripe assay has since been used to uncover guidance cues and mechanisms in many principal neuronal projections, including, for example, the hippocampal, thalamic and olfactory systems [8][9][10][11] . The stripe assay was also applied to investigate neurite outgrowth and guidance behavior related to neuronal regeneration 12-15 and branch formation 16 . Furthermore, the stripe assay has proven valuable in dissecting signaling cascades initiated downstream of axonal guidance receptors activated by ligands printed in a striped pattern [12][13][14][15] . In addition, the guidance properties of membrane-tethered molecules as well as the short-range action of secreted guidance molecules (such as Slits and Semaphorins) could be mimicked in vitro by this assay 17,18 . Also stripe assay experiments were conducted to unravel potential guidance properties of extracellular matrix components 19,20 and of cell adhesion molecules of the IgG superfamily 21 .Of note, the stripe assay was employed as an in vitro assay to investigate the navigation responses of both axonal growth cones toward guidance cues and of migrating cells 4,22,23 . With respect to the latter, neural crest cells 22 and oligodendrocytes 23,24 , but also non-neuronal cells such as tumor cells, have been studied 25 .The assay allows assessment of guidance activity of established molecules and also testing potential guidance properties of hitherto uncharacterized molecules such as RGM, the Wnt signaling inhibitor SFRP1 or the morphogen Sonic h...

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Analysis of Cell‐Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules

et al. 2013

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Cell‐cell adhesion is a fundamental requirement for all multicellular organisms. The calcium‐independent cell adhesion molecules of the immunoglobulin superfamily (IgSF‐CAMs) represent a major subgroup. They consist of immunoglobulin folds alone or in combination with other protein modules, often fibronectin type‐III folds. More than 100 IgSF‐CAMs have been identified in vertebrates and invertebrates. Most of the IgSF‐CAMs are cell surface molecules that are membrane‐anchored either by a single transmembrane segment or by a glycosylphosphatidylinositol (GPI) anchor. Some of the IgSF‐CAMs also occur in soluble form, e.g., in the cerebrospinal fluid or in the vitreous fluid of the eye, due to naturally occurring cleavage of the GPI anchor or the membrane‐proximal peptide segment. Some IgSF‐CAMs, such as NCAM, occur in various forms that are generated by alternative splicing. This unit contains a series of protocols that have been used to study the function of IgSF‐CAMs in vitro and in vivo. Curr. Protoc. Cell Biol. 61:9.5.1‐9.5.85. © 2013 by John Wiley & Sons, Inc.

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Microcontact printing of axon guidance molecules for generation of graded patterns

Cited by 134 publications

References 23 publications

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

Laminin-332 coordinates mechanotransduction and growth cone bifurcation in sensory neurons

Stripe assay to examine axonal guidance and cell migration

Analysis of Cell‐Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules

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