Correction: Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties.

Correction for 'Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties' by Jaclyn Raeburn et al., Soft Matter, 2012, 8, 1168-1174, https://doi.org/10.1039/C1SM06929B.

FtsZ treadmilling is essential for Z-ring condensation and septal constriction initiation in Bacillus subtilis cell division.

Despite the central role of division in bacterial physiology, how division proteins work together as a nanoscale machine to divide the cell remains poorly understood. Cell division by cell wall synthesis proteins is guided by the cytoskeleton protein FtsZ, which assembles at mid-cell as a dense Z-ring formed of treadmilling filaments. However, although FtsZ treadmilling is essential for cell division, the function of FtsZ treadmilling remains unclear. Here, we systematically resolve the function of FtsZ treadmilling across each stage of division in the Gram-positive model organism Bacillus subtilis using a combination of nanofabrication, advanced microscopy, and microfluidics to measure the division-protein dynamics in live cells with ultrahigh sensitivity. We find that FtsZ treadmilling has two essential functions: mediating condensation of diffuse FtsZ filaments into a dense Z-ring, and initiating constriction by guiding septal cell wall synthesis. After constriction initiation, FtsZ treadmilling has a dispensable function in accelerating septal constriction rate. Our results show that FtsZ treadmilling is critical for assembling and initiating the bacterial cell division machine.

Movement dynamics of divisome proteins and PBP2x:FtsW in cells of Streptococcus pneumoniae.

Bacterial cell division and peptidoglycan (PG) synthesis are orchestrated by the coordinated dynamic movement of essential protein complexes. Recent studies show that bidirectional treadmilling of FtsZ filaments/bundles is tightly coupled to and limiting for both septal PG synthesis and septum closure in some bacteria, but not in others. Here we report the dynamics of FtsZ movement leading to septal and equatorial ring formation in the ovoid-shaped pathogen, Streptococcus pneumoniae Conventional and single-molecule total internal reflection fluorescence microscopy (TIRFm) showed that nascent rings of FtsZ and its anchoring and stabilizing proteins FtsA and EzrA move out from mature septal rings coincident with MapZ rings early in cell division. This mode of continuous nascent ring movement contrasts with a failsafe streaming mechanism of FtsZ/FtsA/EzrA observed in a ΔmapZ mutant and another Streptococcus species. This analysis also provides several parameters of FtsZ treadmilling in nascent and mature rings, including treadmilling velocity in wild-type cells and ftsZ(GTPase) mutants, lifetimes of FtsZ subunits in filaments and of entire FtsZ filaments/bundles, and the processivity length of treadmilling of FtsZ filament/bundles. In addition, we delineated the motion of the septal PBP2x transpeptidase and its FtsW glycosyl transferase-binding partner relative to FtsZ treadmilling in S. pneumoniae cells. Five lines of evidence support the conclusion that movement of the bPBP2x:FtsW complex in septa depends on PG synthesis and not on FtsZ treadmilling. Together, these results support a model in which FtsZ dynamics and associations organize and distribute septal PG synthesis, but do not control its rate in S. pneumoniae.

TAT and HA2 facilitate cellular uptake of gold nanoparticles but do not lead to cytosolic localisation.

The methods currently available to deliver functional labels and drugs to the cell cytosol are inefficient and this constitutes a major obstacle to cell biology (delivery of sensors and imaging probes) and therapy (drug access to the cell internal machinery). As cell membranes are impermeable to most molecular cargos, viral peptides have been used to bolster their internalisation through endocytosis and help their release to the cytosol by bursting the endosomal vesicles. However, conflicting results have been reported on the extent of the cytosolic delivery achieved. To evaluate their potential, we used gold nanoparticles as model cargos and systematically assessed how the functionalisation of their surface by either or both of the viral peptides TAT and HA2 influenced their intracellular delivery. We evaluated the number of gold nanoparticles present in cells after internalisation using photothermal microscopy and their subcellular localisation by electron microscopy. While their uptake increased when the TAT and/or HA2 viral peptides were present on their surface, we did not observe a significant cytosolic delivery of the gold nanoparticles.

Photothermal microscopy of the core of dextran-coated iron oxide nanoparticles during cell uptake.

A detailed understanding of cellular interactions with superparamagnetic iron oxide nanoparticles (SPIONs) is critical when their biomedical applications are considered. We demonstrate how photothermal microscopy can be used to follow the cellular uptake of SPIONs by direct imaging of the iron oxide core. This offers two important advantages when compared with current strategies employed to image magnetic cores: first, it is nondestructive and is therefore suitable for studies of live cells and, second, it offers a higher sensitivity and resolution, thus allowing for the identification of low levels of SPIONs within a precise subcellular location. We have shown that this technique may be applied to the imaging of both cell monolayers and cryosections. In the former we have demonstrated the role of temperature on the rate of endocytosis, while in the latter we have been able to identify cells labeled with SPIONs from a mixed population containing predominantly unlabeled cells. Direct imaging of the SPION core is of particular relevance for research involving clinically approved SPIONs, which do not contain fluorescent tags and therefore cannot be detected via fluorescence microscopy.

Fmoc-diphenylalanine hydrogels: understanding the variability in reported mechanical properties.

Fmoc-diphenylalanine (FmocFF or FmocPhePhe) is an important low molecular weight hydrogelator. Gelation can be induced by either lowering the pH of an aqueous solution of FmocFF or by the addition of water to a solution of FmocFF in a solvent such as DMSO. Despite the volume of literature on FmocFF, the mechanical properties reported for the gels vary significantly over four orders of magnitude and the origins of this variability is unclear. Here, we study systematically the mechanical properties of FmocFF gels prepared with different protocols. We demonstrate that the final pH of the gels is the principal determinant of the mechanical properties independently of the method of gel formation. We also show that additional variability arises from experimental factors such as the fraction of DMSO or the nature of the buffers used in selected systems.

Gold nanoparticles delivery in mammalian live cells: a critical review.

Functional nanomaterials have recently attracted strong interest from the biology community, not only as potential drug delivery vehicles or diagnostic tools, but also as optical nanomaterials. This is illustrated by the explosion of publications in the field with more than 2,000 publications in the last 2 years (4,000 papers since 2000; from ISI Web of Knowledge, 'nanoparticle and cell' hit). Such a publication boom in this novel interdisciplinary field has resulted in papers of unequal standard, partly because it is challenging to assemble the required expertise in chemistry, physics, and biology in a single team. As an extreme example, several papers published in physical chemistry journals claim intracellular delivery of nanoparticles, but show pictures of cells that are, to the expert biologist, evidently dead (and therefore permeable). To attain proper cellular applications using nanomaterials, it is critical not only to achieve efficient delivery in healthy cells, but also to control the intracellular availability and the fate of the nanomaterial. This is still an open challenge that will only be met by innovative delivery methods combined with rigorous and quantitative characterization of the uptake and the fate of the nanoparticles. This review mainly focuses on gold nanoparticles and discusses the various approaches to nanoparticle delivery, including surface chemical modifications and several methods used to facilitate cellular uptake and endosomal escape. We will also review the main detection methods and how their optimum use can inform about intracellular localization, efficiency of delivery, and integrity of the surface capping.

Cathepsin L digestion of nanobioconjugates upon endocytosis.

Understanding the dynamic fate and interactions of bioconjugated nanoparticles within living cells and organisms is a prerequisite for their use as in situ sensors or actuators. While recent research has provided indications on the effect of size, shape, and surface properties of nanoparticles on their internalization by living cells, the biochemical fate of the nanoparticles after internalization has been essentially unknown. Here we show that, upon internalization in a wide range of mammalian cells, biological molecules attached to the nanoparticles are degraded within the endosomal compartments through peptide cleavage by the protease cathepsin L. Importantly, using bioinformatics tools, we show that cathepsin L is able to cleave more than a third of the human proteome, indicating that this degradation process is likely to happen to most nanoparticles conjugated with peptides and proteins and cannot be ignored in the design of nanomaterials for intracellular applications. Preservation of the bioconjugates can be achieved by a combination of cathepsin inhibition and endosome disruption.