High-efficacy subcellular micropatterning of proteins using fibrinogen anchors

Abstract

Protein micropatterning allows proteins to be precisely deposited onto a substrate of choice and is now routinely used in cell biology and in vitro reconstitution. However, drawbacks of current technology are that micropatterning efficiency can be variable between proteins and that proteins may lose activity on the micropatterns. Here, we describe a general method to enable micropatterning of virtually any protein at high specificity and homogeneity while maintaining its activity. Our method is based on an anchor that micropatterns well, fibrinogen, which we functionalized to bind to common purification tags. This enhances micropatterning on various substrates, facilitates multiplexed micropatterning, and dramatically improves the on-pattern activity of fragile proteins like molecular motors. Furthermore, it enhances the micropatterning of hard-to-micropattern cells. Last, this method enables subcellular micropatterning, whereby complex micropatterns simultaneously control cell shape and the distribution of transmembrane receptors within that cell. Altogether, these results open new avenues for cell biology.

Subcellular micropatterning for visual immunoprecipitation reveals differences in cytosolic protein complexes downstream the EGFR

“…Compared to the control conditions, we detected a ~4-fold increase in bait protein enrichment in antibody patterned areas. This value is comparable to other studies using different protein patterning approaches (Singhai et al, 2014; Dirscherl et al, 2018; Watson et al, 2021), again proofing the effective surface functionalization of our platform.…”

“…Protein micropatterning can lead to the formation of protein clusters within or at the cell membrane with subsequent recruitment of relevant proteins (Watson et al, 2021), including bait and prey molecules of interest (Lanzerstorfer et al, 2020). To investigate unspecific bait-prey copatterning in the presented approach, bait-PAR and prey distribution was checked on microstructured surfaces but without antibody incubation ( Figure S1 ).…”

“…Most recently, a similar approach for real-time quantification of cytosolic PPIs using cell-based molography as a biosensor was developed (Incaviglia et al, 2020). In addition, subcellular micropatterning of artificial transmembrane receptors was proofed by using fibrinogen anchors (Watson et al, 2021).…”

Engineering the Cellular Microenvironment of Post-infarct Myocardium on a Chip

“…The cardiac myocytes were aligned using microcontact printing, which involves the preparation of silicone “stamps” that are used to transfer extracellular matrix proteins to a substrate in desired geometries. With a spatial resolution of approximately 1 μm, microcontact printing can direct tissue orientation ( 82 – 84 ), single-cell shape ( 85 ), and even subcellular structures ( 86 ). When the aligned cardiac tissues contracted on the cantilever substrates, the cantilevers deflected, leading to a resistance change in the embedded strain sensors proportional to the contractile stress ( 87 , 88 ).…”

Easy and robust micropatterning using fibrinogen anchors

“…Further, the authors take advantage of the library of different fibrinogen conjugates to generate patterns containing three different proteins that bind to three different sequentially patterned fibrinogen conjugates. While the improvements in selectivity and homogeneity achieved by Watson et al are alone a significant step forward, the most exciting demonstrations in their study is that their method enables maintenance of the biological activity of printed proteins in a number of different contexts (6). For example, the authors pattern proteins to perform a microtubule gliding assay and show that printed kinesin motors maintain their ability to move microtubules with the expected gliding speed and dynamics.…”