title: Magnetoskeleton : Elucidating cytoskeletal elements involved in magnetosome chain assembly and segregation of Magnetospirillum gryphiswaldense
Creators: name: Toro-Nahuelpan, Mauricio
Publisher: name: University of Bayreuth
Year: 2018
Subjects: subject: magnetotactic bacteria
subject: magnetotaxis
subject: magnetosomes
subject: cytoskeleton
subject: cell cycle
subject: cell division
subject: segregation
subject: partitioning
subject: magnetoskeleton
subject: actin-like
subject: MamK
subject: MamY
subject: MipZ
subject: PopZ
subject: cryo-electron tomography
subject: cell biology
subject: organelles
subject: 500
subject: 570

description: Magnetotactic bacteria synthesize dedicated magnetic organelles termed magnetosomes, which are membrane-enveloped magnetic nanocrystals. To serve as an efficient magnetoreceptor, magnetosomes are organized into a single, midcell positioned and tightly spaced chain that aligns the cell to the Earth’s magnetic field, facilitating taxis towards a suitable environment. In the model organism Magnetospirillum gryphiswaldense, the magnetosome chain is assembled by concerted action of the actin-like MamK filament and the adaptor protein MamJ, which links the magnetosomes to the MamK cytoskeleton. To pass on the selective advantage of magnetotaxis to ensure cell fitness, during cytokinesis, the magnetosome chain is regularly found traversing the cell division site, where it becomes equipartitioned. Subsequently, the newly divided chains must undergo a hypothesized dynamic relocalization to midcell, which has been attributed to MamK. Yet, the underling molecular mechanisms of magnetosomes segregation, putative dynamics and coordination with the cell cycle have remained elusive. In this thesis, in vivo time-lapse imaging and photokinetic approaches revealed that a directed MamK filament treadmilling is responsible for a rapid and dynamic pole-to-midcell repositioning of magnetosome chains in recently divided cells. In addition, it was found that magnetosomes equal partitioning is highly precise and directly dependent on an intact MamK filament dynamics. Thus, MamK dynamics and its interplay with MamJ provide an active driving force for magnetosomes movement. Peculiarly, loss of MamK did not abolish magnetosome chain configuration, but merely caused fragmented chains, revealing an additional yet unrecognized determinant for magnetosomes organization. In this work, a subcellular analysis by cryo-electron tomography (cryo-ET) showed that the magnetosome chain is confined to the positive curvature of the cytoplasmic membrane of the helical cell (i.e., the geodetic axis). Interestingly, the chain geodetic position became lost in absence of the transmembrane protein MamY. Moreover, double deletion of mamY and mamK entirely abolished chain configuration, suggesting MamY as a novel and essential scaffolding element for magnetosomes organization and positioning. This notion was substantiated by construction of an artificial magnetosome chain on MamY, circumventing both MamK and MamJ functions. PALM and 3D-SIM analyses demonstrated that MamY is highly enriched along the geodetic axis, likely in a polymerized state. These results evidence that MamY acts as a landmark of positive cell curvature and positions a linear magnetoreceptor inside a helical cell, aligning the motility axis and magnetosome chain magnetic-dipole moment. Therefore, MamY is proposed as a new member of the hereby-termed magnetoskeleton, which comprises essential proteins for magnetosomes spatial organization. Since coordination of magnetosome dynamics with cell cycle is yet poorly understood, the role of two major cell cycle regulators, PopZ and MipZ, was analyzed in this context. In Alphaproteobacteria, PopZ serves as a polar landmark that anchors the chromosomes to the cell poles allowing faithful segregation and cell division, whereas MipZ is an essential protein that acts downstream of PopZ and forms an intracellular gradient for proper septum localization. M. gryphiswaldense possesses two MipZ homologs from which only MipZ-like 1 displayed a gradient-like localization. Unexpectedly, single and double deletion of the mipZ-like homologs was feasible, a major difference with the previously reported essential MipZ function. However, only the absence of MipZ-like 1 impaired cell division. Similarly, deletion of a popZ homolog elicited cell filamentation and polar mini-cells. Although, the three homologs were not found to influence magnetosomes or MamK dynamics, PopZ and MipZ-like 1 proved to be key players involved in cell cycle control of M. gryphiswaldense, which, interestingly, appears to have a slightly distinct cell division regulation. In addition, PopZ has been previously suggested to assemble a polar filamentous scaffold, yet direct evidence was missing. In this thesis, Volta phase plate (VPP) cryo-ET showed that PopZ generates a 3D network of disordered, flexible, tightly packed and cross-linked filaments at the cell poles of M. gryphiswaldense. This direct imaging of PopZ filaments confirmed the existence of a genuine bacterial cytoskeletal network in situ. During the course of this work, extensive cryo-ET analyses were performed and led to the unforeseen discovery of novel magnetosomal and subcellular structures. VPP cryo-ET enabled visualization of a periplasmic plug-like structure associated with nascent magnetosomes, which could possibly correspond to the long-speculated barrier between the lumen of the vesicle and the periplasmic space. In addition, a ribosome-free exclusion zone around magnetosomes and a surfacial concentric cover-like structure were detected, likely corresponding to a magnetosomal protein shell. Furthermore, several other unidentified cellular structures were observed and briefly summarized. Altogether, the results of this thesis demonstrate that the magnetoskeleton and the cell cycle in M.gryphiswaldense are more complex than previously assumed and that magnetosomes represent one of the most intricate levels of organelle organization known in bacteria. Finally, this work converges and substantiates the missive that bacteria are complex, powerful and fascinating forms of life.