Protocol for precision editing of endogenous Chlamydomonas reinhardtii genes with CRISPR-Cas.

CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line.

A high-bandwidth voltage amplifier for driving piezoelectric actuators in high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) is a technique capable of revealing the dynamics of biomolecules and living organisms at the nanoscale with a remarkable temporal resolution. The phase delay in the feedback loop dictates the achievable speed of HS-AFM instruments that rely on fast nanopositioners operated predominantly in conjunction with piezoelectric actuators (PEAs). The high capacitance and high operating voltage of PEAs make them difficult to drive.

Efficient precision editing of endogenous genes with CRISPR-Cas.

CRISPR-Cas genome engineering in the unicellular green algal model has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line.

Conversion of anterograde into retrograde trains is an intrinsic property of intraflagellar transport.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).

Time-Resolved Scanning Ion Conductance Microscopy for Three-Dimensional Tracking of Nanoscale Cell Surface Dynamics.

Nanocharacterization plays a vital role in understanding the complex nanoscale organization of cells and organelles. Understanding cellular function requires high-resolution information about how the cellular structures evolve over time. A number of techniques exist to resolve static nanoscale structure of cells in great detail (super-resolution optical microscopy, EM, AFM).

Kinetic and structural roles for the surface in guiding SAS-6 self-assembly to direct centriole architecture.

Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole.

Overlapping and essential roles for molecular and mechanical mechanisms in mycobacterial cell division.

Mechanisms to control cell division are essential for cell proliferation and survival . Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes to maintain mechanical integrity of the cell wall . Recent studies suggest that cell separation is governed by mechanical forces .

Towards a mechanistic understanding of cellular processes by cryoEM.

A series of recent hardware and software developments have transformed cryo-electron microscopy (cryoEM) from a niche tool into a method that has become indispensable in structural and functional biology. Samples that are rapidly frozen are encased in a near-native state inside a layer of amorphous ice, and then imaged in an electron microscope cooled to cryogenic temperatures. Despite being conceptually simple, cryoEM owns its success to a plethora of technological developments from numerous research groups.

Single-molecule kinetics of pore assembly by the membrane attack complex.

The membrane attack complex (MAC) is a hetero-oligomeric protein assembly that kills pathogens by perforating their cell envelopes. The MAC is formed by sequential assembly of soluble complement proteins C5b, C6, C7, C8 and C9, but little is known about the rate-limiting steps in this process. Here, we use rapid atomic force microscopy (AFM) imaging to show that MAC proteins oligomerize within the membrane, unlike structurally homologous bacterial pore-forming toxins.

Detecting topological variations of DNA at single-molecule level.

In addition to their use in DNA sequencing, ultrathin nanopore membranes have potential applications in detecting topological variations in deoxyribonucleic acid (DNA). This is due to the fact that when topologically edited DNA molecules, driven by electrophoretic forces, translocate through a narrow orifice, transient residings of edited segments inside the orifice modulate the ionic flow. Here we utilize two programmable barcoding methods based on base-pairing, namely forming a gap in dsDNA and creating protrusion sites in ssDNA for generating a hybrid DNA complex.

Photothermal Off-Resonance Tapping for Rapid and Gentle Atomic Force Imaging of Live Cells.

Imaging living cells by atomic force microscopy (AFM) promises not only high-resolution topographical data, but additionally, mechanical contrast, both of which are not obtainable with other microscopy techniques. Such imaging is however challenging, as cells need to be measured with low interaction forces to prevent either deformation or detachment from the surface. Off-resonance modes which periodically probe the surface have been shown to be advantageous, as they provide excellent force control combined with large amplitudes, which help reduce lateral force interactions.

Time-Resolved Imaging of Bacterial Surfaces Using Atomic Force Microscopy.

Time-resolved atomic force microscopy (AFM) offers countless new modes by which to study bacterial cell physiology on relevant time scales, from mere milliseconds to hours and days on end. In addition, time-lapse AFM acts as a complementary tool to optical fluorescence microscopy (OFM), for which the combination offers a correlative link between the physical manifestation of bacterial phenotypes and molecular mechanisms obeying those principles. Herein we describe the essential materials and methods necessary for conducting time-resolved AFM and dual AFM/OFM experiments on bacteria.

High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6.

The self-assembly of protein complexes is at the core of many fundamental biological processes, ranging from the polymerization of cytoskeletal elements, such as microtubules, to viral capsid formation and organelle assembly. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement.

Digitally controlled analog proportional-integral-derivative (PID) controller for high-speed scanning probe microscopy.

Nearly all scanning probe microscopes (SPMs) contain a feedback controller, which is used to move the scanner in the direction of the z-axis in order to maintain a constant setpoint based on the tip-sample interaction. The most frequently used feedback controller in SPMs is the proportional-integral (PI) controller. The bandwidth of the PI controller presents one of the speed limiting factors in high-speed SPMs, where higher bandwidths enable faster scanning speeds and higher imaging resolution.

Division site selection linked to inherited cell surface wave troughs in mycobacteria.

Cell division is tightly controlled in space and time to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation. It is unknown how division site selection is controlled in bacteria that lack homologues of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections.

Harnessing the damping properties of materials for high-speed atomic force microscopy.

The success of high-speed atomic force microscopy in imaging molecular motors, enzymes and microbes in liquid environments suggests that the technique could be of significant value in a variety of areas of nanotechnology. However, the majority of atomic force microscopy experiments are performed in air, and the tapping-mode detection speed of current high-speed cantilevers is an order of magnitude lower in air than in liquids. Traditional approaches to increasing the imaging rate of atomic force microscopy have involved reducing the size of the cantilever, but further reductions in size will require a fundamental change in the detection method of the microscope.

Studying biological membranes with extended range high-speed atomic force microscopy.

High-speed atomic force microscopy has proven to be a valuable tool for the study of biomolecular systems at the nanoscale. Expanding its application to larger biological specimens such as membranes or cells has, however, proven difficult, often requiring fundamental changes in the AFM instrument. Here we show a way to utilize conventional AFM instrumentation with minor alterations to perform high-speed AFM imaging with a large scan range.

High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy.

Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging.

High-frequency multimodal atomic force microscopy.

Multifrequency atomic force microscopy imaging has been recently demonstrated as a powerful technique for quickly obtaining information about the mechanical properties of a sample. Combining this development with recent gains in imaging speed through small cantilevers holds the promise of a convenient, high-speed method for obtaining nanoscale topography as well as mechanical properties. Nevertheless, instrument bandwidth limitations on cantilever excitation and readout have restricted the ability of multifrequency techniques to fully benefit from small cantilevers.

High-speed imaging upgrade for a standard sample scanning atomic force microscope using small cantilevers.

We present an atomic force microscope (AFM) head for optical beam deflection on small cantilevers. Our AFM head is designed to be small in size, easily integrated into a commercial AFM system, and has a modular architecture facilitating exchange of the optical and electronic assemblies. We present two different designs for both the optical beam deflection and the electronic readout systems, and evaluate their performance.

A high frequency sensor for optical beam deflection atomic force microscopy.

We demonstrate a novel electronic readout for quadrant photodiode based optical beam deflection setups. In our readout, the signals used to calculate the deflections remain as currents, instead of undergoing an immediate conversion to voltages. Bipolar current mirrors are used to perform all mathematical operations at the transistor level, including the signal normalizing division.