CELL CYCLE PALM, Final Report
The rise of antibiotic resistance represents a grave challenge for mankind. With no new classes of
antibiotics discovered since 1987, an improved understanding of bacterial cell biology will be crucial
to identifying and studying novel antibiotic targets. Furthermore, bacteria represent an excellent
model system for fundamental research, due to their genetic tractability and compatibility with highthroughput multiplexing.
During the period of this FP7 project, we used the new technique of super-resolution fluorescence
microscopy to study bacterial cell biology. This combination is extremely exciting, since it allows one
of the key challenges to studying living bacteria – their small size (a few microns) – to be overcome,
revealing the nanoscale organization of key bacterial proteins.
Our major innovation was to conceive of and build a high-throughput super-resolution imaging
modality that would allow us to study hundreds of cells in a single experiment (Fig. 1). This allowed
us to study the cell-cycle dependent organization of the essential cell division protein FtsZ, a key
next-generation antibiotic target. We found that FtsZ forms a patchy “band” at mid-cell (Fig. 2-3),
and not a continuous ring as previously thought (Holden et al., PNAS 2014). This challenges the “FtsZcentric” model of cell division, suggesting that FtsZ probably does not generate constrictive force,
and may merely recruit other proteins to the division site. This has significant implications for our
understanding of cell division, and will motivate further research into alternative physical
mechanisms for constriction.
The project generated multiple significant publications. In addition to a PNAS paper on FtsZ
cytokinesis (Holden et al., PNAS 2014), the project also lead to or supported publications on
nanoscale analysis of bacterial transcription (Endesfelder et al, Biophys. J 2013), and high speed in
vivo super-resolution imaging (Min et al. Sci. Rep. 2014, Min et al. Biomed. Opt. Expr. 2014). We have
also released several pieces of software related to the project, which are linked to from
http://leb.epfl.ch/software.
Outreach was performed by blogging (http://seamusholden.wordpress.com/), release of Youtube
videos to explain the results (see website), and liasing with press (HTPALM was featured in Nature
Methods and Microscopy & Microanalysis.
The new results and techniques developed during the project will support further investigation and
understanding of medically relevant processes in bacterial cell biology, especially bacterial cell
division. This new findings will inform and support the development of new antibiotics targeting
these processes.
Figure 1: HTPALM schematic. A. i-ii. PALM and phase contrast (PH) images are automatically acquired for many
fields of view, as the cell cycle progresses in a synchronized bacterial population. Fields of view acquired later in
the experiment contain bacteria in later stages of the cell cycle. iii. Single molecule localizations are extracted
from the PALM images, and cell outlines are extracted from the PH images. iv. Data is combined to produce a
HTPALM time lapse showing super-resolved changes in single molecule localization over a whole cell cycle. B.
HTPALM is facilitated by (i) a high-stability home-built microscope, which significantly reduces drift , (ii) closedloop control of the density of bright fluorophores, and (iii) custom software to automatically search for bacteria
and record PALM and PH images. From Holden et al., PNAS 2014.
Figure 2: FtsZ predominantly forms patches or incomplete rings. i-v. 3D volume reconstruction of mid-cell FtsZ
localization for 5 separate bacteria in the early pre-divisional stage of cell cycle. Complete rings (v) are much
less common than the other morphologies shown. From Holden et al., PNAS 2014.
Figure 3: Model of Z-ring organization, including new information from HTPALM measurements. i. During the
stalked cell-cycle stage, the Z-ring assembles at mid-cell. Sparsely distributed non-interacting protofilaments
are randomly distributed around the circumference of the inner membrane, forming a patchy band. ii. As the
cell cycle progresses (late PD), Z-ring radius decreases. Reduced radius means randomly distributed
protofilaments are more likely to overlap circumferentially. iii. At the end of the late pre-divisional stage, a
period of rapid Z-ring contraction occurs, followed by scission. From Holden et al., PNAS 2014.