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Divalent cations behave as effective cross-linkers of intermediate filaments (IFs) such as vimentin IF (VIF). These interactions have been mostly attributed to their multivalency. However, ion-protein interactions often depend on the ion species, and these effects have not been widely studied in IFs. Here, we investigate the effects of two biologically important divalent cations, Zn 2þ and Ca 2þ , on VIF network structure and mechanics in vitro. We find that the network structure is unperturbed at micromolar Zn 2þ concentrations, but strong bundle formation is observed at a concentration of 100 mM. Microrheological measurements show that network stiffness increases with cation concentration. However, bundling of filaments softens the network. This trend also holds for VIF networks formed in the presence of Ca 2þ , but remarkably, a concentration of Ca 2þ that is two orders higher is needed to achieve the same effect as with Zn 2þ , which suggests the importance of salt-protein interactions as described by the Hofmeister effect. Furthermore, we find evidence of competitive binding between the two divalent ion species. Hence, specific interactions between VIFs and divalent cations are likely to be an important mechanism by which cells can control their cytoplasmic mechanics.

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be introduced, which is mainly achieved by adding synthetic DNA barcode primers to beads. For these applications, the beads are mostly made of biocompatible polymers, for example, polyacrylamide beads in inDrop, [2] hydroxylated methacrylic polymer beads used in Drop-seq, [3] and polyacrylamide beads used in 10X Genomics. [8] Each of these chemically different beads has their own advantages and shortcomings. The inDrop polyacrylamide bead can be closely packed in a microfluidic device channel to achieve more than 95% loading of single bead per drop. [2] However, in the inDrop system, UV light is necessary to release primers from the bead, which somewhat complicates bead fabrication and makes it less cost effective. [9] Another shortcoming is that UV light may introduce damage to DNA or RNA and skew the results. [10,11] The Drop-seq system does not release primers from the bead and the reaction efficiency is low as reactions happen only near the surface of beads. [3] In the 10X Genomics platform, gel beads can be efficiently delivered into drops, but the comparably high cost and lack of flexibility in designing Droplet-based single cell sequencing technologies, such as inDrop, Dropseq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.

Significance Filamentous actin (F-actin) and vimentin intermediate filaments (VIFs) are two major cytoskeletal components; they are generally thought to be spatially compartmentalized and to have distinctly different and independent functions. Here we combine two imaging methods, high-resolution structured illumination microscopy and cryo-electron tomography, as well as functional characterizations, to show that unexpectedly, VIFs and F-actin have extensive structural interactions within the cell cortex and form interpenetrating networks. These interactions have very important functional consequences for cells, which are broadly significant given the wide range of processes attributed to F-actin. These results profoundly alter our understanding of the contributions of cytoskeletal components and counter the common belief that VIFs and F-actin are independent in both structure and function.