Coiled coil proteins in bacterial cell architecture
How is it possible to build a three-dimensional cell based on the linear information contained in the genes?
This is a task all organisms face. The cytoskeleton, a network of dynamic protein filaments, is an important tool to structurally organize the cells both in eukaryotes and prokaryotes. In animal cells one of the major cytoskeletal systems is called the intermediate filaments (IF). IFs are composed of coiled coil proteins and their typical function is to strengthen the physical properties of the cells, and help to sense and withstand mechanical stress. Over 30 human diseases, such as progeria and skin blistering disease, to name a few, have been linked to mutations in genes encoding IF proteins. Despite their importance, IFs are the least understood components of the eukaryotic cytoskeleton. This is due to the difficulties to crystallize IF proteins for structure determination, and due to the lack of powerful genetic systems for functional studies. We and others have shown that an analogous cytoskeleton is also present in bacteria, making them attractive as tractable model organisms to study the structure and functions of IF-like cytoskeletons.
Crescentin in Caulobacter crescentus
Crescentin is the first bacterial protein that was recognized as an IF protein. Previously IFs were considered as being a strictly metazoan property. Crescentin has IF-like protein structure, biochemical properties and cytoskeletal function. Crescentin forms a filamentous structure along one lateral side (green in figure) and bends the otherwise straight Caulobacter crescentus cell into a moon crescent-like shape.
Fluorescence microscopy image showing crescentin-GFP (green; Cres-GFP) in the inner curvature of Caulobacter crescentus cells (stained red). Wildtype cells (wt) have a moon crescent-like shape, whereas cells without crescentin (Δcres) are straight rods (grey images are DIC micrographs).
Coiled coil cytoskeleton of Streptomyces
Coiled coil proteins are emerging as main determinants of morphology and polarity inStreptomyces, which is a tractable model system to study a large and important group of actinobacteria. Similarly with eukaryotic systems, such as filamentous fungi, pollen tubes and root hairs,Streptomyces hyphae exhibit polar growth, which is one of the most extreme manifestations of cellular polarity. Streptomyces species often colonize soil and have a complicated lifestyle resembling that of filamentous fungi. The vegetative mycelium grows by elongation of the tips of the branching hyphae, until nutrient depletion triggers an elaborate differentiation process, which culminates in secondary metabolism production and sporulation. In Streptomyces a coiled coil protein DivIVA is responsible for establishment and maintenance of cell polarity. In addition to DivIVA we have found that FilP, a protein with IF-like cytoskeletal properties, is involved in polar growth. FilP possesses a coiled coil rod domain, which enables it to polymerize into a cytoskeletal network in Streptomyces cells. Direct measurements with atomic force microscopy showed that the FilP structures were needed for proper rigidity and elasticity of the cells, a function similar to that of the IF cytoskeleton in metazoa. Consequently, filP deletion hyphae exhibited distorted morphology, especially when grown on solid surfaces. Our most recent finding is that there is a dynamic interplay between the polarity determinant DivIVA and the IF-like cytoskeleton of FilP.
Overlay of fluorescence and phase contrast micrographs showing hyphae of a wildtypeStreptomyces coelicolor strain. FilP cytoskeleton is shown in green.
Structural basis of the IF-like cytoskeleton
We believe that that the universal biological functions of coiled coil proteins in eukaryotes, bacteria and archaea are based on the physical properties of long coiled coils, such as elasticity, flexibility and mechanical strength. How and why do the coiled coil proteins self-assemble into regular filamentous structures? What are the properties of these structures?
FilP spontaneously assembles into regular striated filaments in vitro after denaturation and subsequent renaturation into Tris-buffers with neutral pH. The partitioned scale bar in the lower left hand corner corresponds to 200 nm.