In Vitro Interaction of the Housekeeping SecA1 with the Accessory SecA2 Protein of Mycobacterium tuberculosis

The majority of proteins that are secreted across the bacterial cytoplasmic membrane leave the cell via the Sec pathway, which in its minimal form consists of the dimeric ATP-driven motor protein SecA that associates with the protein-conducting membrane pore SecYEG. Some Gram-positive bacteria contain two homologues of SecA, termed SecA1 and SecA2. SecA1 is the essential housekeeping protein, whereas SecA2 is not essential but is involved in the translocation of a subset of proteins, including various virulence factors. Some SecA2 containing bacteria also harbor a homologous SecY2 protein that may form a separate translocase. Interestingly, mycobacteria contain only one SecY protein and thus both SecA1 and SecA2 are required to interact with SecYEG, either individually or together as a heterodimer. In order to address whether SecA1 and SecA2 cooperate during secretion of SecA2 dependent proteins, we examined the oligomeric state of SecA1 and SecA2 of Mycobacterium tuberculosis and their interactions with SecA2 and the cognate SecA1, respectively. We conclude that both SecA1 and SecA2 individually form homodimers in solution but when both proteins are present simultaneously, they form dissociable heterodimers

Characterization of the annular lipid shell of the Sec translocon

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids

Design and validation of a human brain endothelial microvessel-on-a-chip open microfluidic model enabling advanced optical imaging

We describe here the design and implementation of an in vitro microvascular open model system using human brain microvascular endothelial cells. The design has several advantages over other traditional closed microfluidic platforms: (1) it enables controlled unidirectional flow of media at physiological rates to support vascular function, (2) it allows for very small volumes which makes the device ideal for studies involving biotherapeutics, (3) it is amenable for multiple high resolution imaging modalities such as transmission electron microscopy (TEM), 3D live fluorescence imaging using traditional spinning disk confocal microscopy, and advanced lattice light sheet microscopy (LLSM). Importantly, we miniaturized the design, so it can fit within the physical constraints of LLSM, with the objective to study physiology in live cells at subcellular level. We validated barrier function of our brain microvessel-on-a-chip by measuring permeability of fluorescent dextran and a human monoclonal antibody. One potential application is to investigate mechanisms of transcytosis across the brain microvessel-like barrier of fluorescently-tagged biologics, viruses or nanoparticles

Exploiting open source 3D printer architecture for laboratory robotics to automate high-throughput time-lapse imaging for analytical microbiology

Growth in open-source hardware designs combined with the low-cost of high performance optoelectronic and robotics components has supported a resurgence of in-house custom lab equipment development. We describe a low cost (below USD700), open-source, fully customizable high-throughput imaging system for analytical microbiology applications. The system comprises a Raspberry Pi camera mounted on an aluminium extrusion frame with 3D-printed joints controlled by an Arduino microcontroller running open-source Repetier Host Firmware. The camera position is controlled by simple G-code scripts supplied from a Raspberry Pi singleboard computer and allow customized time-lapse imaging of microdevices over a large imaging area. Open-source OctoPrint software allows remote access and control. This simple yet effective design allows high-throughput microbiology testing in multiple formats including formats for bacterial motility, colony growth, microtitre plates and microfluidic devices termed ‘lab-on-a-comb’ to screen the effects of different culture media components and antibiotics on bacterial growth. The open-source robot design allows customization of the size of the imaging area; the current design has an imaging area of ~420 × 300mm, which allows 29 ‘lab-on-a-comb’ devices to be imaged which is equivalent 3480 individual 1μl samples. The system can also be modified for fluorescence detection using LED and emission filters embedded on the PiCam for more sensitive detection of bacterial growth using fluorescent dyes

SecA, a remarkable nanomachine

Biological cells harbor a variety of molecular machines that carry out mechanical work at the nanoscale. One of these nanomachines is the bacterial motor protein SecA which translocates secretory proteins through the protein-conducting membrane channel SecYEG. SecA converts chemically stored energy in the form of ATP into a mechanical force to drive polypeptide transport through SecYEG and across the cytoplasmic membrane. In order to accommodate a translocating polypeptide chain and to release transmembrane segments of membrane proteins into the lipid bilayer, SecYEG needs to open its central channel and the lateral gate. Recent crystal structures provide a detailed insight into the rearrangements required for channel opening. Here, we review our current understanding of the mode of operation of the SecA motor protein in concert with the dynamic SecYEG channel. We conclude with a new model for SecA-mediated protein translocation that unifies previous conflicting data

Characterization of the annular lipid shell of the Sec translocon

The bacterial Sec translocase in its minimal form consists of a membrane-embedded protein-conducting pore SecYEG that interacts with the motor protein SecA to mediate the translocation of secretory proteins. In addition, the SecYEG translocon interacts with the accessory SecDFyajC membrane complex and the membrane protein insertase YidC. To examine the composition of the native lipid environment in the vicinity of the SecYEG complex and its impact on translocation activity, styrene-maleic acid lipid particles (SMALPs) were used to extract SecYEG with its lipid environment directly from native Escherichia coli membranes without the use of detergents. This allowed the co-extraction of SecYEG in complex with SecA, but not with SecDFyajC or YidC. Lipid analysis of the SecYEG-SMALPs revealed an enrichment of negatively charged lipids in the vicinity of SecYEG, which in detergent assisted reconstitution of the Sec translocase are crucial for the translocation activity. Such lipid enrichment was not found with separately extracted SecDFyajC or YidC, which demonstrates a specific interaction between SecYEG and negatively charged lipids.</p

A single copy of SecYEG is sufficient for preprotein translocation

The heterotrimeric SecYEG complex comprises a protein-conducting channel in the bacterial cytoplasmic membrane. SecYEG functions together with the motor protein SecA in preprotein translocation. Here, we have addressed the functional oligomeric state of SecYEG when actively engaged in preprotein translocation. We reconstituted functional SecYEG complexes labelled with fluorescent markers into giant unilamellar vesicles at a natively low density. Förster’s resonance energy transfer and fluorescence (cross-) correlation spectroscopy with single-molecule sensitivity allowed for independent observations of the SecYEG and preprotein dynamics, as well as complex formation. In the presence of ATP and SecA up to 80% of the SecYEG complexes were loaded with a preprotein translocation intermediate. Neither the interaction with SecA nor preprotein translocation resulted in the formation of SecYEG oligomers, whereas such oligomers can be detected when enforced by crosslinking. These data imply that the SecYEG monomer is sufficient to form a functional translocon in the lipid membrane.

Membrane-on-a-Chip:Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin a-hemolysin (alpha HL). The influence of lipid composition and ionic strength on aHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. l Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers