RESUMEN
The plant endoplasmic reticulum forms a network of tubules connected by three-way junctions or sheet-like cisternae. Although the network is three-dimensional, in many plant cells, it is constrained to thin volume sandwiched between the vacuole and plasma membrane, effectively restricting it to a 2-D planar network. The structure of the network, and the morphology of the tubules and cisternae can be automatically extracted following intensity-independent edge-enhancement and various segmentation techniques to give an initial pixel-based skeleton, which is then converted to a graph representation. ER dynamics can be determined using optical flow techniques from computer vision or persistency analysis. Collectively, this approach yields a wealth of quantitative metrics for ER structure and can be used to describe the effects of pharmacological treatments or genetic manipulation. The software is publicly available.
Asunto(s)
Benchmarking , Retículo Endoplásmico , Membrana Celular , Alimentos , Células VegetalesRESUMEN
The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.