Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system.

Fluorescent protein tags are fundamental tools used to visualize gene products and analyze their dynamics in vivo. Recent advances in genome editing have expedited the precise insertion of fluorescent protein tags into the genomes of diverse organisms. These advances expand the potential of in vivo imaging experiments and facilitate experimentation with new, bright, photostable fluorescent proteins. Most quantitative comparisons of the brightness and photostability of different fluorescent proteins have been made in vitro, removed from biological variables that govern their performance in cells or organisms. To address the gap, we quantitatively assessed fluorescent protein properties in vivo in an animal model system. We generated transgenic Caenorhabditis elegans strains expressing green, yellow, or red fluorescent proteins in embryos and imaged embryos expressing different fluorescent proteins under the same conditions for direct comparison. We found that mNeonGreen was not as bright in vivo as predicted based on in vitro data but is a better tag than GFP for specific kinds of experiments, and we report on optimal red fluorescent proteins. These results identify ideal fluorescent proteins for imaging in vivo in C. elegans embryos and suggest good candidate fluorescent proteins to test in other animal model systems for in vivo imaging experiments.

Processive acceleration of actin barbed-end assembly by N-WASP.

Neuronal Wiskott-Aldrich syndrome protein (N-WASP)-activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3, N-WASP binds actin-filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We use nanofibers coated with N-WASP WWCA domains as model cell surfaces and single-actin-filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed-end assembly. Individual barbed ends captured by WWCA domains grow at or below their diffusion-limited assembly rate. At high filament densities, however, overlapping filaments form buckles between their nanofiber tethers and myosin attachment points. These buckles grew ∼3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native polyproline (PP) region show similar rate enhancements in the absence of profilin, but profilin slows barbed-end acceleration from constructs containing the PP region. Increasing Mg(2+) to enhance filament bundling increases the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed-end bundling. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion.

Cell isolation and culture.

Cell isolation and culture are essential tools for the study of cell function. Isolated cells grown under controlled conditions can be manipulated and imaged at a level of resolution that is not possible in whole animals or even tissue explants. Recent advances have allowed for large-scale isolation and culture of primary C. elegans cells from both embryos and all four larval stages. Isolated cells can be used for single-cell profiling, electrophysiology, and high-resolution microscopy to assay cell autonomous development and behavior. This chapter describes protocols for the isolation and culture of C. elegans embryonic and larval stage cells. Our protocols describe isolation of embryonic and L1 stage cells from nematodes grown on high-density NA22 bacterial plates and isolation of L2 through L4 stage cells from nematodes grown in axenic liquid culture. Both embryonic and larval cells can be isolated from nematode populations within 3 hours and can be cultured for several days. A primer on sterile cell culture techniques is given in the appendices.

Actin filament attachments for sustained motility in vitro are maintained by filament bundling.

We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.

Isolation and culture of larval cells from C. elegans.

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×10(4) cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology.

Single molecule kinetic analysis of actin filament capping. Polyphosphoinositides do not dissociate capping proteins.

We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 x 10(6) M(-1) s(-1) and dissociation of 0.0003 s(-1) agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), PI(4,5)P(2), and PI(3,4,5)P(3), prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 x 10(5) M(-1) s(-1)) and a faster dissociation rate constant (0.004 s(-1)). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.

Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast.

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy.

Understanding the mechanism of actin polymerization and its regulation by associated proteins requires an assay to monitor polymerization dynamics and filament topology simultaneously. The only assay meeting these criteria is total internal reflection fluorescence microscopy (Amann and Pollard, 2001; Fujiwara et al., 2002). The fluorescence signal is fourfold stronger with actin labeled on Cys-374 with Oregon green rather than rhodamine. To distinguish growth at barbed and pointed ends we used image drift correction and maximum intensity projections to reveal points where single N-ethylmaleimide inactivated myosins attach filaments to the glass coverslip. We estimated association rates at high actin concentrations and dissociation rates near and below the critical actin concentration. At the barbed end, the association rate constant for Mg-ATP-actin is 7.4 microM(-1) s(-1) and the dissociation rate constant is 0.89 s(-1). At the pointed end the association and dissociation rate constants are 0.56 microM(-1) s(-1) and 0.19 s(-1). When vitamin D binding protein sequesters all free monomers, ADP-actin dissociates from barbed ends at 1.4 s(-1) and from pointed ends at 0.16 s(-1) regardless of buffer nucleotide.

The bacterial cytoskeleton: an intermediate filament-like function in cell shape.

Various cell shapes are encountered in the prokaryotic world, but how they are achieved is poorly understood. Intermediate filaments (IFs) of the eukaryotic cytoskeleton play an important role in cell shape in higher organisms. No such filaments have been found in prokaryotes. Here, we describe a bacterial equivalent to IF proteins, named crescentin, whose cytoskeletal function is required for the vibrioid and helical shapes of Caulobacter crescentus. Without crescentin, the cells adopt a straight-rod morphology. Crescentin has characteristic features of IF proteins including the ability to assemble into filaments in vitro without energy or cofactor requirements. In vivo, crescentin forms a helical structure that colocalizes with the inner cell curvatures beneath the cytoplasmic membrane. We propose that IF-like filaments of crescentin assemble into a helical structure, which by applying its geometry to the cell, generates a vibrioid or helical cell shape depending on the length of the cell.

Spatial and temporal pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis.

Microscopy of fluorescent fusion proteins and genetic dependencies show that fission yeast assemble and constrict a cytokinetic contractile ring in a precisely timed, sequential order. More than 90 min prior to separation of the spindle pole bodies (SPB), the anillin-like protein (Mid1p) migrates from the nucleus and specifies a broad band of cortex around the equator as the division site. Between 10 min before and 2 min after SPB separation, conventional myosin-II (Myo2p), IQGAP (Rng2p), PCH protein (Cdc15p), and formin (Cdc12p) join the broad band independent of actin filaments. Over the subsequent 10 min prior to anaphase B, this broad band of proteins condenses into a contractile ring including actin, tropomyosin (Cdc8p), and alpha-actinin (Ain1p). During anaphase B, unconventional myosin-II (Myp2p) joins the ring followed by the septin (Spn1p). Ring contraction and disassembly begin 37 min after SPB separation. This spatial and temporal hierarchy provides the framework for analysis of molecular mechanisms.

The fission yeast cytokinesis formin Cdc12p is a barbed end actin filament capping protein gated by profilin.

Cytokinesis in most eukaryotes requires the assembly and contraction of a ring of actin filaments and myosin II. The fission yeast Schizosaccharomyces pombe requires the formin Cdc12p and profilin (Cdc3p) early in the assembly of the contractile ring. The proline-rich formin homology (FH) 1 domain binds profilin, and the FH2 domain binds actin. Expression of a construct consisting of the Cdc12 FH1 and FH2 domains complements a conditional mutant of Cdc12 at the restrictive temperature, but arrests cells at the permissive temperature. Cells overexpressing Cdc12(FH1FH2)p stop growing with excessive actin cables but no contractile rings. Like capping protein, purified Cdc12(FH1FH2)p caps the barbed end of actin filaments, preventing subunit addition and dissociation, inhibits end to end annealing of filaments, and nucleates filaments that grow exclusively from their pointed ends. The maximum yield is one filament pointed end per six formin polypeptides. Profilins that bind both actin and poly-l-proline inhibit nucleation by Cdc12(FH1FH2)p, but polymerization of monomeric actin is faster, because the filaments grow from their barbed ends at the same rate as uncapped filaments. On the other hand, Cdc12(FH1FH2)p blocks annealing even in the presence of profilin. Thus, formins are profilin-gated barbed end capping proteins with the ability to initiate actin filaments from actin monomers bound to profilin. These properties explain why contractile ring assembly requires both formin and profilin and why viability depends on the ability of profilin to bind both actin and poly-l-proline.

Dynamic polarization of the microtubule cytoskeleton during CTL-mediated killing.

Efficient unidirectional killing by cytotoxic T lymphocytes (CTL) requires translocation of the microtubule organizing center (MTOC) to the target cell contact site. Here we utilize modulated polarization microscopy and computerized 3D reconstruction of tubulin and LFA-1 immunofluorescence images to investigate how this is accomplished. The results show that the MTOC is drawn vectorially to the contact site by a microtubule sliding mechanism. Once the MTOC arrives at the contact site, it oscillates laterally. Microtubules loop through and anchor to a ring-shaped zone (pSMAC) defined by the dense clustering of LFA-1 at the target contact site. Microtubules that run straight between the MTOC and pSMAC and then turn sharply may indicate the action of a microtubule motor such as dynein.