Majid Malboubi

Fascin Regulates Nuclear Movement and Deformation in Migrating Cells

Summary Fascin is an F-actin-bundling protein shown to stabilize filopodia and regulate adhesion dynamics in migrating cells, and its expression is correlated with poor prognosis and increased metastatic potential in a number of cancers. Here, we identified the nuclear envelope protein nesprin-2 as a binding partner for fascin in a range of cell types in vitro and in vivo. Nesprin-2 interacts with fascin through a direct, F-actin-independent interaction, and this binding is distinct and separable from a role for fascin within filopodia at the cell periphery. Moreover, disrupting the interaction between fascin and nesprin-2 C-terminal domain leads to specific defects in F-actin coupling to the nuclear envelope, nuclear movement, and the ability of cells to deform their nucleus to invade through confined spaces. Together, our results uncover a role for fascin that operates independently of filopodia assembly to promote efficient cell migration and invasion.

An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments

Graphical abstract

Surface properties of glass micropipettes and their effect on biological studies

In this paper, an investigation on surface properties of glass micropipettes and their effect on biological applications is reported. Pipettes were pulled under different pulling conditions and the effect of each pulling parameter was analyzed. SEM stereoscopic technique was used to reveal the surface roughness properties of pipette tip and pipette inner wall in 3D. More than 20 pipettes were reconstructed. Pipette heads were split open using focused ion beam (FIB) milling for access to the inner walls. It is found that surface roughness parameters are strongly related on the tip size. Bigger pipettes have higher average surface roughness and lower developed interfacial area ratio. Furthermore, the autocorrelation of roughness model of the inner surface shows that the inner surface does not have any tendency of orientation and is not affected by pulling direction. To investigate the effect of surface roughness properties on biological applications, patch-clamping tests were carried out by conventional and FIB-polished pipettes. The results of the experiments show that polished pipettes make significantly better seals. The results of this work are of important reference value for achieving pipettes with desired surface properties and can be used to explain biological phenomenon such as giga-seal formation.

Study of the Tip Surface Morphology of Glass Micropipettes and Its Effects on Giga-Seal Formation

Reported is a study of applying nanofabrication technology to improve the surface roughness of micro glass pipettes to achieve giga ohm seal resistance in patch clamping processes. The surface roughness of pipette tips was first measured by 3D reconstruction of pipette tips using stereo imaging technique based on high resolution SEM images. Both the SEM images and the reconstructed images show that micro glass pipettes have rough and uneven tips which could be one of the causes of leakage in patch clamping. Then focused ion beam system was used to cut across the very end of the tip, producing a smooth and flat new tip. The average surface area roughness Sa of a milled pipette tip was within a few nanometres. Patch clamping experiments were carried out using the polished pipettes on human umbilical vein endothelial cells (HUVEC), which were well known for their extremely flat shape making them very difficult to patch. The results show that above 3 GΩ seals were achieved in 60% of the experiments, as opposed to \(1.5 - 2.0\,\mathrm{G}\Omega \) in average with the conventional pipettes. The highest seal resistance achieved with a focused ion beam polished pipette was 9 GΩ, well above the 3 GΩ resistance, the usually best result achieved with a conventional pipette. The research results demonstrate that the surface roughness of a pipette has a significant effect on the giga-seal formation of a patch clamping process.

Effect of Tip Size on Gigaseal Formation

This chapter reports the research into the effect of tip size on gigaseal formation. The approach adopted in the research includes, first, investigation on how the tip size affects the patch clamping, then a study on the surface properties of pipettes with different tip sizes and compare the seal resistances obtained by them, and finally verification of the conclusions using experiments.

Development of Patch Clamping

Patch clamping was first introduced into biophysical studies by Neher and Sakmann [1] in 1976 and was soon expanded to many other fields such as biology and medicine. The technique not only allowed the detection of single-channel currents in biological membranes for the first time but also enabled higher current resolution, direct membrane patch potential control, and physical isolation of membrane patches [2]. The development of the patch clamp method was honoured with a Nobel Prize in 1991.

Study of Glass Micropipettes from Tip Formation to Characterization

In this chapter various aspects of glass micropipettes are studied, including mechanisms of tip formation, tip geometry, and effect of pulling parameters on surface roughness properties of glass micropipettes.

Effect of Roughness on Gigaseal Formation

A rough pipette tip in conventional patch clamping, or patching site in planar patch clamping prevents seal formation. In this chapter the effect of roughness on gigaseal formation is discussed.

Effect of Hydrophilicity on Gigaseal Formation

A membrane has both hydrophilic and hydrophobic components. The exact contribution of these components in seal formation is not clear. Hydrophilicity of the pipette (or patch site in planar patch clamping) is believed to be a prerequisite for gigaseal formation.