High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Opto-combinatorial indexing enables high-content transcriptomics by linking cell images and transcriptomes.

We introduce a simple integrated analysis method that links cellular phenotypic behaviour with single-cell RNA sequencing (scRNA-seq) by utilizing a combination of optical indices from cells and hydrogel beads. With our method, the combinations, referred to as joint colour codes, enable the link via matching the optical combinations measured by conventional epi-fluorescence microscopy with the concatenated DNA molecular barcodes created by cell-hydrogel bead pairs and sequenced by next-generation sequencing. We validated our approach by demonstrating an accurate link between the cell image and scRNA-seq with mixed species experiments, longitudinal cell tagging by electroporation and lipofection, and gene expression analysis. Furthermore, we extended our approach to multiplexed chemical transcriptomics, which enabled us to identify distinct phenotypic behaviours in HeLa cells treated with various concentrations of paclitaxel, and determine the corresponding gene regulation associated with the formation of a multipolar spindle.

Design of Mechanical and Electrical Properties for Multidirectional Control of Microtubules.

Microtubule (MT)-motor systems show promise as nanoscale actuator platforms for performing molecular manipulations in nanobiotechnology and micro total analysis systems. These systems have been demonstrated to exert a variety of functions, including the concentration, transportation, and detection of molecular cargos. Although gliding direction control of MTs is necessary for these applications, most direction control methods are currently conducted using micro/nanofabricated guiding structures and/or flow, magnetic, and electric field forces. These control methods force all MTs to exhibit identical gliding behaviors and destinations. In this chapter, we describe an active multidirectional control method for MT without guiding tracks. The bottom-up molecular design allowed MTs to be guided in designated directions under an electric field in a microfluidic device. By designing the stiffness and surface charge density of MTs, three types of MT (Stiff-MT, Soft-MT, and Charged soft-MT) with different mechanical and electrical properties are prepared. The gliding directions within an electric field are predicted according to the measured stiffness and electrophoretic mobility. Finally, the Stiff-MTs are separated from Soft-MTs and Charged soft-MTs with a microfluidic sorter.

Different motilities of microtubules driven by kinesin-1 and kinesin-14 motors patterned on nanopillars.

Kinesin is a motor protein that plays important roles in a variety of cellular functions. In vivo, multiple kinesin molecules are bound to cargo and work as a team to produce larger forces or higher speeds than a single kinesin. However, the coordination of kinesins remains poorly understood because of the experimental difficulty in controlling the number and arrangement of kinesins, which are considered to affect their coordination. Here, we report that both the number and spacing significantly influence the velocity of microtubules driven by nonprocessive kinesin-14 (Ncd), whereas neither the number nor the spacing changes the velocity in the case of highly processive kinesin-1. This result was realized by the optimum nanopatterning method of kinesins that enables immobilization of a single kinesin on a nanopillar. Our proposed method enables us to study the individual effects of the number and spacing of motors on the collective dynamics of multiple motors.

Transport of microtubules according to the number and spacing of kinesin motors on gold nano-pillars.

Motor proteins function in in vivo ensembles to achieve cargo transport, flagellum motion, and mitotic cell division. Although the cooperativity of multiple motors is indispensable for physiological function, reconstituting the arrangement of motors in vitro is challenging, so detailed analysis of the functions of motor ensembles has not yet been achieved. Here, we developed an assay platform to study the motility of microtubules driven by a defined number of kinesin motors spaced in a definite manner. Gold (Au) nano-pillar arrays were fabricated on a silicon/silicon dioxide (Si/SiO2) substrate with spacings of 100 nm to 500 nm. The thiol-polyethylene glycol (PEG)-biotin self-assembled monolayer (SAM) and silane-PEG-CH3 SAM were then selectively formed on the pillars and SiO2 surface, respectively. This allowed for both immobilization of kinesin molecules on Au nano-pillars in a precise manner and repulsion of kinesins from the SiO2 surface. Using arrayed kinesin motors, we report that motor number and spacing do not influence the motility of microtubules driven by kinesin-1 motors. This assay platform is applicable to all kinds of biotinylated motors, allows the study of the effects of motor number and spacing, and is expected to reveal novel behaviors of motor proteins.