Abstract
Biomaterial substrates can be engineered to present topographical signals to cells which, through interactions between the material and active components of the cell membrane, regulate key cellular processes and guide cell fate decisions. However, targeting mechanoresponsive elements that reside within the intracellular domain is a concept that has only recently emerged. Here, we show that mesoporous silicon nanoneedle arrays interact simultaneously with the cell membrane, cytoskeleton, and nucleus of primary human cells, generating distinct responses at each of these cellular compartments. Specifically, nanoneedles inhibit focal adhesion maturation at the membrane, reduce tension in the cytoskeleton, and lead to remodeling of the nuclear envelope at sites of impingement. The combined changes in actin cytoskeleton assembly, expression and segregation of the nuclear lamina, and localization of Yes-associated protein (YAP) correlate differently from what is canonically observed upon stimulation at the cell membrane, revealing that biophysical cues directed to the intracellular space can generate heretofore unobserved mechanosensory responses. These findings highlight the ability of nanoneedles to study and direct the phenotype of large cell populations simultaneously, through biophysical interactions with multiple mechanoresponsive components.
Highlights
Physical cues from the extracellular space are sensed at the cell membrane and initiate intracellular signaling cascades that influence cell fate and function.[1−5] The rational design of materials that are employed as culture substrates enables investigation of how cells respond to physicochemical stimuli from the extracellular matrix (ECM)
Arrays of high aspect ratio, vertically oriented nanostructures have recently garnered tremendous attention for their interactions with the intracellular component of cells in culture and tissues. These materials can deliver membrane-impermeant cargo to the cytosol,[27−34] sense enzymatic activity,[35,36] and maximum; N = 4). (C) qPCR analysis indicates reduced expression of the Yes-associated protein (YAP) target genes ankyrin repeat domain 1 (ANKRD1) and connective tissue growth factor (CTGF) (N = 4, mean ± SD). (D) Cell spread area and YAP nuclear localization correlate tightly on flat substrates, but correlation is weakened on nN (N = 3). (E) YAP localization following cell treatment with either the actin depolymerizing agent, latrunculin B (LatB), or a small molecule to stimulate actin bundling, lysophosphatidic acid (LPA)
Twenty-five features of cell morphology and actin textures were subsequently compared by linear discriminant analysis (LDA), which revealed that actin homogeneity, a measure of fiber size, reduced greatly on nN whereas protrusions extended farther radially from the nucleus (Figure 1D,E)
Summary
Physical cues from the extracellular space are sensed at the cell membrane and initiate intracellular signaling cascades that influence cell fate and function.[1−5] The rational design of materials that are employed as culture substrates enables investigation of how cells respond to physicochemical stimuli from the extracellular matrix (ECM). How intracellular elements are affected by outside-in, canonical mechanosensing.[17−23] In contrast, techniques such as micropipette aspiration,[24] optical/magnetic tweezers,[25] and atomic force microscopy[26] have been used to directly probe individual organelles without relying upon material-derived cues, demonstrating that direct interaction with mechanosensitive organelles can induce changes in cell behaviors Their low throughput and complex setups limit their investigational and translational potential in more advanced tissue and in vivo models. Cells on nanowires exhibit fewer adhesive structures[2,39−42] and reduced cytoskeletal tension,[2,15,17] alongside alterations to cellular[8,29,43−50] and nuclear morphology.[8,51] these observations have generated a wealth of understanding about the membraneinitiated response to nanowires, there remains an unmet need to understand the nature of the interactions between nanomaterials and the intracellular space, as well as how these events influence mechanosensory pathways
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