Many biotechniques use complementary split-fluorescent protein (sFPs) fragments to visualize protein-protein interactions, image cells by ensemble or single molecule fluorescence microscopy, or assemble nanomaterials and protein superstructures. Yet, the reassembly mechanisms of sFPs, including fragment binding rates, folding, chromophore maturation and overall photophysics remain poorly characterized. Here, we evolved asymmetric and self-complementing green, yellow and cyan sFPs together with their full-length equivalents (flFPs) and described their biochemical and photophysical properties in vitro and in cells. While re-assembled sFPs have spectral properties similar to flFPs, they display slightly reduced quantum yields and fluorescence lifetimes due to a less sturdy β-barrel structure. The complementation of recombinant sFPs expressed in vitro follows a conformational selection mechanism whereby the larger sFP fragments exist in a monomer-dimer equilibrium and only monomers are competent for fluorescence complementation. This bimolecular fragment interaction involves a slow and irreversible binding step, followed by chromophore maturation at a rate similar to that of flFPs. When expressed as fusion tags in cells, sFPs behave as monomers directly activated with synthetic complementary fragments. This study resulted in the development of sFP color variants having improved maturation kinetics, brightness, and photophysics for fluorescence microscopy imaging of cellular processes, including single molecule detection.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery–Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

Emerin is an integral nuclear envelope protein participating in the maintenance of nuclear shape. When mutated or absent, emerin causes X-linked Emery-Dreifuss muscular dystrophy (EDMD). To define how emerin takes parts in molecular scaffolding at the nuclear envelope and helps protect the nucleus against mechanical stress, we established its nanoscale organization using single molecule tracking and super-resolution microscopy. We show that emerin monomers form localized oligomeric nanoclusters stabilized by both lamin A/C and SUN1 LINC complex. Interactions of emerin with nuclear actin and BAF additionally modulate its membrane mobility and its ability to oligomerize. In nuclei subjected to mechanical challenges, the mechanotransducing functions of emerin are coupled to changes in its oligomeric state, and the incremental self-assembly of emerin determines nuclear shape adaptation against forces. We also show that the abnormal nuclear envelope deformations induced by EDMD emerin mutants stem from an improper formation of lamin A/C and LINC complex-stabilized emerin oligomers. These findings place emerin at the center of the molecular processes that regulate nuclear shape remodeling in response to mechanical challenges.

X-linked Emery-Dreifuss muscular dystrophy (EDMD) results from mutations of the nuclear envelope protein emerin, which participates in nuclear mechanotransductions and maintenance of nuclear shape. To better understand the molecular determinants of EDMD, we probed the membrane diffusion and the nanoscale spatial organization of emerin and some of its clinically relevant mutations, using single molecule tracking and super-resolution microscopy. We show that emerin is distributed as monomers and oligomeric clusters at the inner nuclear membrane and that both its diffusion and its oligomeric state are modified by EDMD-associated mutations. Emerin interactions with lamin A/C, nuclear actin and BAF modulate its mobility and its membrane organization. Measurements in nuclei under stress further indicate that the mechanotransducing function of emerin are coupled to its oligomeric state and that defective formation of emerin oligomers result in aberrant nuclear deformation against forces. These findings place emerin at the center of the molecular processes that regulate nuclear shape remodeling in response to mechanical stress.