Probing archaeal cell biology: exploring the use of dyes in the imaging of Sulfolobus cells

Archaea are key players in many critical ecological processes. In comparison to eukaryotes and bacteria, however, our understanding of both the cell biology and diversity of archaea remains limited. While archaea inhabit a wide range of environmental conditions, many species are extremophiles, surviving in extreme temperature, salt or pH conditions, making their cell biology hard to study. Recently, our understanding of archaeal cell biology has been advanced significantly by the advent of live cell imaging in extremis as well as the development of genetic tools to exogenously express fluorescent proteins in some mesophilic archaeal model systems, e.g., Haloferax volcanii. However, for most archaeal species, especially thermophilic species or emerging model systems without well characterized genetic tools, live cell imaging remains dependent on fluorescent chemical probes to label and track the dynamics of living cells. While a wide range of fluorescent stains and markers that label different components of the cell are available commercially, their use has usually been optimized for use in a small number of eukaryotic cell systems. Here we report the successes and failures of the application of membrane, DNA, S-layer and cytoplasm markers in live cell imaging of archaea, as well as the optimization of fixation and immunolabelling approaches. We have applied these markers to the thermoacidophilic archaeon Sulfolobus acidocaldarius, but expect some to work in other archaeal species. Furthermore, those procedures that failed in S. acidocaldarius may still prove useful for imaging archaea that grow at a more neutral pH and/or at a less extreme temperature

The patterned assembly and stepwise Vps4-mediated disassembly of composite ESCRT-III polymers drives archaeal cell division

ESCRT-III family proteins form composite polymers that deform and cut membrane tubes in the context of a wide range of cell biological processes across the tree of life. In reconstituted systems, sequential changes in the composition of ESCRT-III polymers induced by the AAA-adenosine triphosphatase Vps4 have been shown to remodel membranes. However, it is not known how composite ESCRT-III polymers are organized and remodeled in space and time in a cellular context. Taking advantage of the relative simplicity of the ESCRT-III-dependent division system in Sulfolobus acidocaldarius, one of the closest experimentally tractable prokaryotic relatives of eukaryotes, we use super-resolution microscopy, electron microscopy, and computational modeling to show how CdvB/CdvB1/CdvB2 proteins form a precisely patterned composite ESCRT-III division ring, which undergoes stepwise Vps4-dependent disassembly and contracts to cut cells into two. These observations lead us to suggest sequential changes in a patterned composite polymer as a general mechanism of ESCRT-III-dependent membrane remodeling. </p

Probing archaeal cell biology: exploring the use of dyes in the imaging of Sulfolobus cells.

Peer reviewed: TrueAcknowledgements: The authors would like to acknowledge all members of the Baum lab for their input throughout the project. Specifically, we would like to thank Andre Pulschen, Gabriel Tarrason Risa and Fredrik Hurtig for initially establishing the methods used in this paper; Jovan Traparic, Matthew Kenneth and Yin-wei Kuo for their input in method optimization and feedback on the manuscript. We would also like to thank the MRC LMB Light Microscopy facility for technical support.Archaea are key players in many critical ecological processes. In comparison to eukaryotes and bacteria, however, our understanding of both the cell biology and diversity of archaea remains limited. While archaea inhabit a wide range of environmental conditions, many species are extremophiles, surviving in extreme temperature, salt or pH conditions, making their cell biology hard to study. Recently, our understanding of archaeal cell biology has been advanced significantly by the advent of live cell imaging in extremis as well as the development of genetic tools to exogenously express fluorescent proteins in some mesophilic archaeal model systems, e.g., Haloferax volcanii. However, for most archaeal species, especially thermophilic species or emerging model systems without well characterized genetic tools, live cell imaging remains dependent on fluorescent chemical probes to label and track the dynamics of living cells. While a wide range of fluorescent stains and markers that label different components of the cell are available commercially, their use has usually been optimized for use in a small number of eukaryotic cell systems. Here we report the successes and failures of the application of membrane, DNA, S-layer and cytoplasm markers in live cell imaging of archaea, as well as the optimization of fixation and immunolabelling approaches. We have applied these markers to the thermoacidophilic archaeon Sulfolobus acidocaldarius, but expect some to work in other archaeal species. Furthermore, those procedures that failed in S. acidocaldarius may still prove useful for imaging archaea that grow at a more neutral pH and/or at a less extreme temperature

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells.

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism

An endosomal tether undergoes an entropic collapse to bring vesicles together

An early step in intracellular transport is the selective recognition of a vesicle by its appropriate target membrane, a process regulated by Rab GTPases via the recruitment of tethering effectors. Membrane tethering confers higher selectivity and efficiency to membrane fusion than the pairing of SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) alone. Here we address the mechanism whereby a tethered vesicle comes closer towards its target membrane for fusion by reconstituting an endosomal asymmetric tethering machinery consisting of the dimeric coiled-coil protein EEA1 (refs 6, 7) recruited to phosphatidylinositol 3-phosphate membranes and binding vesicles harbouring Rab5. Surprisingly, structural analysis reveals that Rab5:GTP induces an allosteric conformational change in EEA1, from extended to flexible and collapsed. Through dynamic analysis by optical tweezers, we confirm that EEA1 captures a vesicle at a distance corresponding to its extended conformation, and directly measure its flexibility and the forces induced during the tethering reaction. Expression of engineered EEA1 variants defective in the conformational change induce prominent clusters of tethered vesicles in vivo. Our results suggest a new mechanism in which Rab5 induces a change in flexibility of EEA1, generating an entropic collapse force that pulls the captured vesicle towards the target membrane to initiate docking and fusion

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved