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Aug 20, 2009 - Figure 1. Cell adhesion molecule DM-GRASP offered as a nanopatterned substrate to DRG neurons. (a) Schematic of DM-GRASP molecules ...
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NANO LETTERS

Nanopatterns Biofunctionalized with Cell Adhesion Molecule DM-GRASP Offered as Cell Substrate: Spacing Determines Attachment and Differentiation of Neurons

2009 Vol. 9, No. 12 4115-4121

Steffen Jaehrling, Karsten Thelen, Tobias Wolfram, and G. Elisabeth Pollerberg* Department of DeVelopmental Neurobiology, Institute of Zoology, UniVersity of Heidelberg, 69120 Heidelberg, Im Neuenheimer Feld 232, Germany Received July 21, 2009; Revised Manuscript Received August 13, 2009

ABSTRACT The density/spacing of plasma membrane proteins is thought to be crucial for their function; clear-cut experimental evidence, however, is still rare. We examined nanopatterns biofunctionalized with cell adhesion molecule DM-GRASP with respect to their impact on neuron attachment and neurite growth. Data analysis/modeling revealed that these cellular responses improve with increasing DM-GRASP density, with the exception of one spacing which does not allow for the anchorage of a cytoskeletal protein (spectrin) to three DM-GRASP molecules.

Cell adhesion molecules (CAMs) are integral membrane proteins that possess physical as well as instructive functions: they mediate adhesion between cell surfaces and transform environmental information into cytoskeletal (re)organization.1 Some CAMs have been shown to bind to the proteins (spectrin, ankyrin) of the cortical cytoskeleton (located directly beneath the cell membrane) thereby driving their recruitment into this structure; this in turn affects the lateral mobility and local accumulation of the CAMs and thus cell adhesion and neurite formation.2-5 For both propertiessadhesive and instructivesthe CAM density in the cell membrane is of pivotal importance and there is evidence that it underlies tight regulation mechanisms: Cell surface CAM density has been shown to depend on levels of expression, recycling, and degradation.6,7 We could previously show that internalization and degradation of the neural CAM DM-GRASP (dorsal funiculus, ventral midline immunoglobulin-like restricted axonal surface protein)8-10 (Figure 1a) lower its presence in the plasma membrane and affect neurite functions.11 We thus wanted to analyze the impact of DM-GRASP density on cellular properties of neurons, e.g., attachment and differentiation, by a highly defined approach. For this, we confronted neurons prepared from dorsal root ganglia (DRG) with a series of substrates displaying DMGRASP in distinct nanopatterns (Figure 1a) mimicking DM* Corresponding author: phone, +49-6221-546371; fax, +49-6221546375; e-mail, [email protected]. 10.1021/nl9023325 CCC: $40.75 Published on Web 08/20/2009

 2009 American Chemical Society

GRASP trans-interactions as they take place in cell-cell contact sites in a controllable assay system. Hexagonally arranged gold nanodots (distances 29-137 nm) were fabricated by a technique based on the self-assembly of goldloaded diblock copolymer micelles.12-15 The gold nanodots were equipped with Ni2+-NTA groups16 ensuring the C-terminal binding of the histidine-tagged extracellular domain of DM-GRASP which was produced in HEK293 cells and purified by Ni2+-NTA chromatography.17 The space between the gold nanodots was passivated by poly(ethylene glycol) (PEG) to prevent deposition of proteins shed by cells during the culture period.18-20 The selective presence of DM-GRASP in the area covered by the biofunctionalized gold dots was visualized by indirect immunofluorescence labeling of DM-GRASP (Figure 1b). In addition, neuron attachment and neurite formation were also restricted to the DM-GRASP-functionalized gold nanodotcovered area (Figure 1c). Both observations demonstrate the effective PEG passivation. The neurons displayed DMGRASP in the plasma membrane independent of the substrate used, i.e., on DM-GRASP presenting nanopatterns as well as on laminin-coated (Figure 1d), poly-L-lysine (PLL)coated, and plain glass coverslips (not shown). Comparison of DM-GRASP’s immunofluorescence signal of the nanopattern with the one of cell membrane allowed its plasma membrane concentration to be determined. Brightness level quantifications revealed a density of about 1600 DM-GRASP molecules/µm2 in the plasma membrane (see

Figure 1. Cell adhesion molecule DM-GRASP offered as a nanopatterned substrate to DRG neurons. (a) Schematic of DM-GRASP molecules in the plasma membrane with their C-termini (C) reaching into the cortical cytoskeleton trans-interacting with DM-GRASP molecules of the nanopattern. The extracellular domain of DM-GRASP (ec-DM-GRASP, 4.5 nm diameter, 20 nm in length) is coupled to the gold dot (5 nm diameter, yellow) in physiological orientation, i.e., the N-terminus (N) is directed toward the opposing cell membrane. The PEG layer between the gold dots (4.5 nm in height) leaves the monothiol NTA linker (red square) accessible and prevents deposition of proteins. (b) Immunofluorescence staining of ec-DM-GRASP selectively labels the area containing the DM-GRASP-coupled, nanopatterned gold dots and visualizes a defined, straight border (“dipping line”) delineating the nonpatterned, PEG-passivated area. (c) Single cells prepared from chick embryo DRGs were cultured for 24 h on DM-GRASP nanopatterns. Immunofluorescence labeling of DM-GRASP reveals that the neurons exclusively attach to the DM-GRASP nanopattern; neurite extension is also restricted to this area, avoiding the nonpatterned area. (d) DRG neurons cultured on laminin-coated glass express DM-GRASP and form neurites (as they do on PLL-coated and plain glass, not shown) indicating the independence of these processes from DM-GRASP substrate contact.

supplementary Figure 1 in Supporting Information). For the experiments reported here, nanopatterns with dot interdistances from 137 to 29 nm, i.e., DM-GRASP densities from 62 to 1302 molecules/µm2, respectively, were used. These physiological to subphysiological densities thus allow each nanopattern-DM-GRASP to trans-interact with a cell surfaceDM-GRASP. The substrate DM-GRASP can hence be considered to be the limiting factor (and not the plasma membrane DM-GRASP). Those DM-GRASP molecules in the plasma membrane which do not get a trans-interaction partner may either join clusters induced by the transinteracting DM-GRASPs or remain single and hence inactive as has been shown for a closely related CAM;21 clustering of DM-GRASP, moreover, has been shown to be a prerequisite for its proper function.11 The chosen nanodistances therefore ensure that the nanopatterns are accurately imprinted into the plasma membrane, i.e., that the spacing of trans-interacting DM-GRASP molecules in the plasma membrane truly reflects the spacing of the nanopattern. To gain insight into the adhesive strength of DM-GRASP, we compared neuron adhesion on several substrates commonly used for neural cell culture to the one on DM-GRASP (Figure 2a; supplementary Figure 2a in Supporting Information). Single cell cultures prepared from DRGs were sparsely seeded (146 cells/mm2) and cultured on the various substrates for 24 h. Attached neurons were then visualized (by β3tubulin immunofluorescence labeling) and quantified. DMGRASP’s capability to mediate cell attachment (98 neurons/ mm2) ranks between the one of laminin (132 neurons/mm2), 4116

an extracellular matrix protein binding to integrins in the cell membrane of DRG neurons, and PLL (82 neurons/mm2), a positively charged amino acid polymer attaching cells by electrostatic interactions.22,23 Laminin and (to a lesser degree) PLL are among the most effective known mediators of adhesion for neural cells including DRG neurons.24 Also uncoated glass appears to possess a considerable adhesiveness (62 cells/mm2); this can be largely attributed, however, to proteins shed by the DRG cells during the culture period binding to the glass surface and mediating cell adhesion as indicated by the almost complete absence of attached cells (5 neurons/mm2) on PEG-passivated glass. A large variety of proteins capable of mediating cell attachment, including CAMs and extracellular matrix proteins, have been demonstrated to be released by cultured DRGs.25-29 Such proteins released by neural cell cultures have been shown to be deposited on substrate surfaces and to mediate cell attachment.30,31 Thus the effective passivation by PEG, preventing unspecific and uncontrollable protein deposition (see Figure 1b,c), is crucial for gaining unbiased, interpretable data. To study the impact of the concentration of DM-GRASP offered as substrate on cell attachment, coverslips were incubated (for 1 h at 37 °C) with various DM-GRASP solutions (concentrations ranging from 1-50 µg/mL; Figure 2b and supplementary Figure 2b in Supporting Information). DRG cells were seeded on these substrates, cultured, and stained as described above. To monitor the amounts of DMGRASP bound to the glass surfaces, coverslips were immunolabeled (using DM-GRASP antibodies), fluorescence Nano Lett., Vol. 9, No. 12, 2009

Figure 2. Neuron attachment on conventional and nanopatterned substrates. (a) Numbers of neurons attached to the indicated substrates were determined by evaluation of micrographs randomly taken on coverslips (size of optic field, 0.1376 mm2; OF, number of optic fields evaluated; n, number of independent experiments; PEG, poly(ethylene glycol); PLL, poly-L-lysine; DM, DM-GRASP; LN, laminin). (b) Neuron densities (determined as described above) rise with increasing DM-GRASP concentration in the coating solutions (µg/mL) as well as with bound DM-GRASP which is measured by determining DMGRASP immunofluorescence (IF) brightness levels (a.u., arbitrary units) on the coverslips. (c) Neuron densities (determined as described above) decrease with increasing spacing (29, 54, 70, 86, and 137 nm) of nanopatterned DM-GRASP; in addition, the corresponding DMGRASP densities (molecules/µm2) and the IF brightness levels measured on the nanosubstrates are indicated. Note that the graduation of these additional features on the abscissa is not linear. For complete specification of n, OF, and significance values see supplementary Figure 2 in Supporting Information. Nano Lett., Vol. 9, No. 12, 2009

intensities quantified (ImageJ, NIH), and DM-GRASP densities determined (supplemenary Figure 3 in Supporting Information). Coverslips incubated with low concentrations of DM-GRASP (1 and 5 µg/mL corresponding to calculated 2366 and 11832 DM-GRASP molecules/µm2, respectively, under the assumption that 100% of the molecules bound to the glass) displayed only a low density of attached neurons (58 and 64 neurons/mm2) comparable to the one observed on uncoated glass (62 neurons/mm2). Incubation with the double concentration of DM-GRASP (10 µg/mL corresponding to calculated 23664 DM-GRASP molecules/µm2) resulted in a clear increase (+23%) in neuron attachment. Application of even higher concentrated DM-GRASP coating solutions (20 and 50 µg/mL corresponding to calculated 47330 and 118325 DM-GRASP molecules/µm2) caused only minor increases in cell attachment (+5 and +17%, respectively) indicating saturation of the glass surface (see supplementary Figure 3 in Supporting Information). Those substrate DMGRASP densities which promote cell attachment are orders of magnitude higher than the DM-GRASP density in the cell membrane (about 1600 molecules/µm2). The observed enhanced cell attachment in response to increasing densities of substrate DM-GRASP might thus indicate the inaccessibility of a considerable proportion of the substrate-bound DM-GRASP (probably due to clustering and/or random N-C terminal orientation) for trans-interactions with the plasma membrane-resident DM-GRASP. Therefore, despite the possibility to determine the amount of bound protein (by immunofluorescence brightness measurement), the unknown proportion(s) of inaccessible hence inactive protein make the collection of unequivocally interpretable data impossible. To analyze neuron attachment in response to DM-GRASP substrates with defined density, homogeneous spacing, and physiological orientation, we made use of nanopatterns. When control proteins (bovine serum albumin) were coupled to the gold dots, no cell attachment is observed.32 Biofunctionalization with DM-GRASP (Figure 2c and supplementary Figure 2c in Supporting Information) caused a considerable cell attachment (69 neurons/mm2) to the most narrowly spaced patterns (29 nm). With increasing DM-GRASP spacing, numbers of attached neurons drop to very low levels (5 neurons/mm2) in an almost linear fashion, as expected if cellular adhesion was a merely physical phenomenon. On the 70 nm spaced nanopattern, however, neuron attachment barely reaches the level as observed on the 86 nm pattern (which possesses only 65% of the density of DM-GRASP in 70 nm patterns). The underlying reason might be the geometry of the spectrin network (below the cell membrane) being only insufficiently stabilized on 70 nm patterns (see below and supplementary Figure 5 in Supporting Information). To investigate the impact of DM-GRASP on neuronal differentiation in relation to other substrates, we analyzed neurite formation and elongation (Figure 3a,b and supplementary Figure 4a,b in Supporting Information). Nearly all DRG neurons (90%) sent out neurites during the 24 h in culture on laminin, a potent neurite-inducing protein for DRGs and other neurons.24,33,34 DM-GRASP was almost as 4117

Figure 3. Neurite formation and elongation on various substrate molecules. (a) Numbers of neurite-bearing DRG neurons (as a percentage of all neurons) on the indicated substrates were determined by taking randomly distributed micrographs of the cell cultures (size of optic field, 0.1376 mm2; OF, number of optic fields evaluated; n, number of independent experiments; PEG, poly(ethylene glycol); PLL, poly-L-lysine; DM, DM-GRASP; LN, laminin). (b) Neurite lengths were determined on micrographs (as described above) using ImageJ delineating the single neurites. (c) Increasing DM-GRASP densities on the coverslips (measured as DM-GRASP immunofluorescence (IF) brightness levels; a.u., arbitrary units) due to raising DM-GRASP concentrations in the coating solutions (µg/ mL) result in an increasing percentage of neurons to form neurites; low coating concentrations (0, 1, and 5 µg/mL) do not cause any significant increase. Note that the graduation of the concentration on the abscissa is not linear. For complete specification of n, OF, and significance values see supplementary Figure 4a in Supporting Information. 4118

effective in driving neurite formation (73%) as laminin. Also on PLL-coated and on uncoated glass, a substantial number of neurons formed neurites (66 and 57%, respectively) which can be attributed to shed and subsequently substrate-bound proteins, as indicated by the complete absence of neurite formation (0%) on substrates passivated by PEG. Neurite formation has indeed been shown to be induced by proteins shed and deposited in cell cultures.35-40 The lengths the neurites achieve during 24 h on the various culture substrates (Figure 3b and Figure 4b in Supporting Information) is similar on glass, PLL, and DM-GRASP (279, 262, and 252 µm, respectively), indicating similar intrinsic growth properties of the neurites once they are induced to form. On coverslips coated with a high concentration of laminin, neurite length (349 µm) is only moderately increased (by about one-third) compared to the one on DM-GRASP. When cultured on glass coverslips coated with increasing concentrations (1-50 µg/mL) of DM-GRASP (Figure 3c and supplementary Figure 4c in Supporting Information), DRG single cell cultures displayed a concomitantly raising proportion of neurons which form neurites (51-73%). Neuritogenesis on lower concentrations of DM-GRASP (1 and 5 µg/ mL), in contrast, did not differ significantly from the one on plain glass. This is not due to too low DM-GRASP concentrations to be effective (nanopatterns display lower densities of DM-GRASP; see below) but rather to the relatively high levels of neuritogenesis induced by shed and subsequently substrate-bound proteins (see Figure 3a) which can be assumed to mask the relatively subtle effects caused by low DM-GRASP concentrations. DM-GRASP nanopatterns which are PEG-passivated and therefore free of bound, shed proteins reveal that neurite growth clearly correlates to the DM-GRASP density on the substrate. The percentage of neurite forming neurons (Figure 4a and supplementary Figure 6a in Supporting Information) decreases (49 to 0%) with increasing DM-GRASP spacing (29 to 137 nm, corresponding to 1302-62 DM-GRASP molecules/ µm2). Only on 70 nm spaced DM-GRASP nanopatterns, the neuritogenesis rate (20%) is only half of the expected value (about 40% in a continuous decrease). Neurite length (Figure 4b and supplementary Figure 6b in Supporting Information) is not significantly enhanced by increasing DM-GRASP density. As observed for neuron attachment andsto a higher degreesneurite formation, also neurite growth is significantly reduced on 70 nm spaced nanopatterns (see below). Sequence analysis of the intracellular domain of DMGRASP (35 amino acids, 10 positively charged amino acids) revealed a prominent cluster of five positively charged amino acids at the N-terminus which results in an isoelectric point of 9.34 (calculated with EMBL online resource: Gateway to Isoelectric Point Service) for the entire intracellular domain of DM-GRASP. This strongly indicates that DM-GRASP is able to bind FERM (four point one-ezrin-radixin-moesin superfamily) proteins (for review see ref 41) since these linker proteins have been shown to bind to intracellular domains of integral membrane proteins with high isoelectric points (CD44, 8.17; CD43, 9.24; ICAM, 12.9842), which indicates the FERM binding region (no specific amino acid Nano Lett., Vol. 9, No. 12, 2009

Figure 4. (a) Neurite formation (determined as described in Figure 2) decreases with increasing spacing (29, 54, 70, 86, and 137 nm) of nanopatterned DM-GRASP; in addition to the spacing distance, the corresponding DM-GRASP densities (molecules/µm2), and the immunofluorescence (IF) brightness levels measured on the nanosubstrates are indicated. Note that the graduation of these additional features on the abscissa is not linear. (b) Neurite lengths (determined as described in Figure 3) display a clear drop on 70 nm nanopatterns, as observed for neurite formation. For complete specification of n, OF, and significance values see supplementary Figure 6 in Supporting Information. (c) Schematic of the cell surface-nanopattern interphase: DM-GRASP molecules in the plasma membrane trans-interact with DM-GRASP molecules of the nanopattern. The dimensions of the spectrin heterotetramer (green), i.e., its length and the position of binding site (light green) for the FERM member merlin allow for the interaction with DM-GRASP (via merlin (blue) or other FERMs (purple) and F-actin (gray). On all nanopatterns usedsexcept for the 70 and 137 nm spaced onessthese interactions are possible at three sites on the spectrin heterotetramer (see supplementary Figures 5 and 6 in Supporting Information).

motif). FERM proteins could thus link DM-GRASP to spectrin, both indirectly by virtue of their actin binding site (radixin, moesin43) and directly by their spectrin binding site (merlin44) (Figure 4c and supplementary Figure 6 in Supporting Information). In cells on DM-GRASP nanopatterns with 29, 54, and 86 nm spacing, each spectrin heterotetramer (length 160-180 nm; see supplementary Figure 5.2 in Supporting Information) can be linked at three binding sites (at both ends and in the middle of the tetramer) to DM-GRASP molecules in the plasma membrane which trans-interact with DM-GRASP bound to gold dots (Figure 4c). In contrast, on 70 nm spaced patterns, maximally two linkages (at both ends or at one end and in the middle) of the spectrin heterotetramer to transinteracting DM-GRASP molecules are possible. On the 137 nm spaced pattern, only a single linkage (at the end or in the middle of spectrin) can take place (supplementary Figure 6 in Supporting Information). The decreased number of linkages (one or two) of the spectrin tetramer to the transNano Lett., Vol. 9, No. 12, 2009

interacting DM-GRASP molecules on the 70/137 nm patterns could explain for the reduced/lost cell attachment and neurite formation. Only the trans-interacting DM-GRASP molecules connect spectrin to the nanopattern; lack/reduction of this substrate anchorage is likely to reduce the stability of the spectrin lattice and thus the entire cortical cytoskeleton, leading to an impaired cell attachment. Linkage of spectrin to the cell membrane has been shown to be crucial for proper cortical cytoskeleton architecture and cell attachment.45-47 DMGRASP’s capability to mediate cell adhesion and its motility has been shown to depend on an intact cortical cytoskeleton,48 predominantly composed of short actin filaments and spectrin. Moreover, binding of spectrin (via ankyrin) to the CAM L1 could be demonstrated to be crucial for cell attachment mediated by homophilic trans-interaction.49 Spectrin and several CAMs have been shown to be highly enriched at cell-cell contact sites.4,50-53 For NCAM and a close relative 4119

of DM-GRASP (Lu/B-CAM), direct spectrin interaction could be demonstrated.54,55 Interestingly, the reduction of the cellular responses on the 70 nm spaced patterns (compared to 54 nm pattern) is much more pronounced with respect to neurite formation (-60%) than to neuron attachment (-30%). In contrast to the mere adhesion of a soma, outgrowth of a neurite tip requires a highly organized and stabilized cortical cytoskeleton of which spectrin is a key component since traction forces have to be generated. Spectrin, which is ubiquitously present in all neurons and is enriched in the most adhesive regions of neurites, their tips,56 has been shown to be crucial for neurite formation and extension.57-60 Also members of the FERM family, which have been found in DRG neurons as well as other neuron types and are probably present in all neurons,61-63 have been shown to be required for neurite formation and elongation.62,64-66 The spectrin heterotetramers possess several direct and indirect (via FERMs, actin, and ankyrin) possibilities to connect to a large number of integral plasma membrane proteins. On DM-GRASP nanopattern substrates, those plasma membrane proteins which are able to trans-interact with DM-GRASP molecules of the substrate and thus to be stabilized are known, i.e., DM-GRASP molecules. Nanopattern substratessin addition preventing other types of trans-interaction due to effective passivations hence allow for selective analysis of specific transmembrane protein subpopulations. We here present for the first time a study revealing a significantly reduced cellular response at a specific spacing (70 nm) of a nanopattern. This reduction in cell attachment and neurite formation is independent of the overall density as it “recovers” on 86 nm patterns. Instead, matching of the DM-GRASP-spectrin connections with the distances on the nanopatterns revealed that the formation of a well-stabilized spectrin network is not possible on 70 nm nanopattern. By use of conventional coverslips coated with DM-GRASP, such insights are not possible due to random protein spacing, an unknown percentage of inaccessible proteins, and conformational changes due to uncontrolled adsorption to glass which is a poorly understood process.67-70 In contrast, nanopatterns provide the unique possibility to exert an impact with nanometer accuracy on plasma membrane protein organization and thereby cortical cytoskeleton architecture, depending on the dimensions of the studied molecule. Moreover, the distance-specific effects of the nanopatterned substrates on neurite length indicates the potential of nanopatterns for fine-tuning of neurite growth, e.g., for neurite regeneration. Acknowledgment. The project is funded by the German Research Council (DFG; GK791, PO289, and SFB488). We are grateful to M. Kapp, M. Zieher-Lorenz, C. Brandel, and S. Bergmann for excellent technical assistance. Supporting Information Available: Experimental details, complete specification of number of experiments and significance values, and expanded discussions including schematic drawings. This material is available free of charge via the Internet at http://pubs.acs.org. 4120

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