The Archaeal Cell Cycle.

Since first identified as a separate domain of life in the 1970s, it has become clear that archaea differ profoundly from both eukaryotes and bacteria. In this review, we look across the archaeal domain and discuss the diverse mechanisms by which archaea control cell cycle progression, DNA replication, and cell division. While the molecular and cellular processes archaea use to govern these critical cell biological processes often differ markedly from those described in bacteria and eukaryotes, there are also striking similarities that highlight both unique and common principles of cell cycle control across the different domains of life. Since much of the eukaryotic cell cycle machinery has its origins in archaea, exploration of the mechanisms of archaeal cell division also promises to illuminate the evolution of the eukaryotic cell cycle.

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.

Design centering enables robustness screening of pattern formation models.

MOTIVATION: Access to unprecedented amounts of quantitative biological data allows us to build and test biochemically accurate reaction-diffusion models of intracellular processes. However, any increase in model complexity increases the number of unknown parameters and, thus, the computational cost of model analysis. To efficiently characterize the behavior and robustness of models with many unknown parameters remains, therefore, a key challenge in systems biology. RESULTS: We propose a novel computational framework for efficient high-dimensional parameter space characterization of reaction-diffusion models in systems biology. The method leverages the Lp-Adaptation algorithm, an adaptive-proposal statistical method for approximate design centering and robustness estimation. Our approach is based on an oracle function, which predicts for any given point in parameter space whether the model fulfills given specifications. We propose specific oracles to efficiently predict four characteristics of Turing-type reaction-diffusion models: bistability, instability, capability of spontaneous pattern formation and capability of pattern maintenance. We benchmark the method and demonstrate that it enables global exploration of a model's ability to undergo pattern-forming instabilities and to quantify robustness for model selection in polynomial time with dimensionality. We present an application of the framework to pattern formation on the endosomal membrane by the small GTPase Rab5 and its effectors, and we propose molecular mechanisms underlying this system. AVAILABILITY AND IMPLEMENTATION: Our code is implemented in MATLAB and is available as open source under https://git.mpi-cbg.de/mosaic/software/black-box-optimization/rd-parameter-space-screening. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A non-linear system patterns Rab5 GTPase on the membrane.

Proteins can self-organize into spatial patterns via non-linear dynamic interactions on cellular membranes. Modelling and simulations have shown that small GTPases can generate patterns by coupling guanine nucleotide exchange factors (GEF) to effectors, generating a positive feedback of GTPase activation and membrane recruitment. Here, we reconstituted the patterning of the small GTPase Rab5 and its GEF/effector complex Rabex5/Rabaptin5 on supported lipid bilayers. We demonstrate a 'handover' of Rab5 from Rabex5 to Rabaptin5 upon nucleotide exchange. A minimal system consisting of Rab5, RabGDI and a complex of full length Rabex5/Rabaptin5 was necessary to pattern Rab5 into membrane domains. Rab5 patterning required a lipid membrane composition mimicking that of early endosomes, with PI(3)P enhancing membrane recruitment of Rab5 and acyl chain packing being critical for domain formation. The prevalence of GEF/effector coupling in nature suggests a possible universal system for small GTPase patterning involving both protein and lipid interactions.

Auto-regulation of Rab5 GEF activity in Rabex5 by allosteric structural changes, catalytic core dynamics and ubiquitin binding.

Intracellular trafficking depends on the function of Rab GTPases, whose activation is regulated by guanine exchange factors (GEFs). The Rab5 GEF, Rabex5, was previously proposed to be auto-inhibited by its C-terminus. Here, we studied full-length Rabex5 and Rabaptin5 proteins as well as domain deletion Rabex5 mutants using hydrogen deuterium exchange mass spectrometry. We generated a structural model of Rabex5, using chemical cross-linking mass spectrometry and integrative modeling techniques. By correlating structural changes with nucleotide exchange activity for each construct, we uncovered new auto-regulatory roles for the ubiquitin binding domains and the Linker connecting those domains to the catalytic core of Rabex5. We further provide evidence that enhanced dynamics in the catalytic core are linked to catalysis. Our results suggest a more complex auto-regulation mechanism than previously thought and imply that ubiquitin binding serves not only to position Rabex5 but to also control its Rab5 GEF activity through allosteric structural alterations.

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.

WIPI ß-propellers at the crossroads of autophagosome and lipid droplet dynamics.

Macroautophagy (autophagy hereafter) is an evolutionarily highly conserved catabolic process activated by eukaryotes in order to counteract cellular starvation. Autophagy leads to bulk degradation of cytoplasmic content in the lysosomal compartment, thereby clearing the cytoplasm and generating nutrients and energy. Upon autophagy initiation, cytoplasmic material becomes sequestered in newly formed double-membrane vesicles termed 'autophagosomes' that subsequently acquire acidic hydrolases for content destruction. The de novo biogenesis of autophagosomes often occurs at the endoplasmic reticulum (ER) and, in many cases, in close proximity to lipid droplets (LDs), intracellular neutral lipid storage reservoirs. LDs are targets of autophagic destruction, but have recently also been shown to contribute to autophagosome formation. In fact, some autophagy-related (Atg) proteins, such as microtubule-associated protein light chain 3 (LC3), Atg2 and Atg14L, functionally contribute to both LD and autophagosome biogenesis. In the present paper, we discuss Atg proteins, including members of the human WD-repeat protein interacting with phosphoinositides (WIPI) family that co-localize prominently with LC3, Atg2 and Atg14L to conceivably integrate LD and autophagosome dynamics.

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.