Characterization of 3D Printed Stretching Devices for Imaging Force Transmission in Live-Cells

Abstract

Introduction-Cell stretch is a method which can rapidly apply mechanical force through cell-matrix and cell-cell adhesions and can be utilized to better understand underlying biophysical questions related to intracellular force transmission and mechanotransduction. Methods-3D printable stretching devices suitable for livecell fluorescent imaging were designed using finite element modeling and validated experimentally. These devices were then used along with FRET based nesprin-2G force sensitive biosensors as well as live cell fluorescent staining to understand how the nucleus responds to externally applied mechanical force in cells with both intact LINC (linker of nucleoskeleton and cytoskeleton) complex and cells with the LINC complex disrupted using expression of dominant negative KASH protein. Results-The devices were shown to provide a larger strain ranges (300% uniaxial and 60% biaxial) than currently available commercial or academic designs we are aware of. Under uniaxial deformation, the deformation of the nucleus of NIH 3T3 cells per unit of imposed cell strain was shown to be approximately 50% higher in control cells compared to cells with a disrupted LINC complex. Under biaxial deformation, MDCK II cells showed permanent changes in the nuclear morphology as well as actin organization upon unloading, indicating that failure, plastic deformation, or remodeling of the cytoskeleton is occurring in response to the applied stretch. Conclusion-Development and open distribution of low-cost, 3D-printable uniaxial and biaxial cell stretching devices compatible with live-cell fluorescent imaging allows a wider range of researchers to investigate mechanical influences on biological questions with only a minimal investment of resources.

Design of a 3D printed, motorized, uniaxial cell stretcher for microscopic and biochemical analysis of mechanotransduction

“…Previous studies have shown that mechanical stretching leads to changes in nuclear shape and size ( Anno et al, 2012 ; Brosig et al, 2010 ; Hoffman et al, 2020 ; Mayer et al, 2019 ). To verify that our stretcher could be used to study the impact of stretching on cells, we subjected U2OS cells to incremental static stretch and measured changes in nuclear area.…”

“…Some are commercially available (Flexcell, Strex, Electron Microscope Sciences, etc. ; Callaghan and Williams, 2000 ; Matsugaki, Fujiwara, and Nakano, 2013 ; Richard et al, 2007 ), and many labs have designed and built their own custom stretchers ( Boulter et al, 2020 ; Daulagala et al, 2020 ; Huang and Nam-Trung, 2013 ; Huang et al, 2010 ; Isermann et al, 2012 ; Mayer et al, 2019 ; Roth et al, 2015 ; Shiwarski et al, 2020 ). Our system contributes to this line-up of stretchers in that it has a very simple design, with easy adaptation to experimental needs, and relatively low cost of entry.…”

“…These stretchers are inexpensive to manufacture and can be used for microscopy and biochemical analysis. Stretching is performed by manually adjusting a cog or screw that changes the strain on the membrane ( Daulagala et al, 2020 ; Mayer et al, 2019 ), or by adding a weight to extend the membrane ( Isermann et al, 2012 ). These stretchers can be used for biaxial ( Isermann et al, 2012 ; Mayer et al, 2019 ) or uniaxial stretching ( Daulagala et al, 2020 ; Mayer et al, 2019 ).…”

The SUN1 splicing variants SUN1_888 and SUN1_916 differentially regulate nucleolar structure

“…Next, to confirm whether interactions between nesprins and SUNs are required for the formation of proper nucleolar morphology, we used a dominant‐negative KASH construct (DN‐KASH, Mayer et al, 2019). Expression of DN‐KASH altered the morphology of the nucleoli and severely decreased the intensity of nucleolin staining compared with untransfected cells in the same field (Figure 3g, arrows indicate transfected cells), demonstrating that the interactions between nesprins and SUNs are indeed required for the determination of nucleolar structure.…”

“…pEGFPN3‐SUN1_888 and pEGFPN3‐SUN1_916 have been previously described (Nishioka et al, 2016). An expression vector for dominant‐negative KASH (DN‐KASH)‐mCherry fusion protein was a gift from Daniel Conway (Virginia Commonwealth University, Addgene plasmid #125553) (Mayer et al, 2019). Expression plasmids for myc‐tagged mouse SUN1_913 and GFP‐tagged SUN1_888(mut) were from OriGene (Rockville, MD, USA) and VectorBuilder, Japan (Kanagawa, Japan), respectively.…”