Daniel P. Mulvihill

The role of Plo1 kinase in mitotic commitment and septation in Schizosaccharomyces pombe

Plo1‐associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M‐phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1Δ cells to septate and the prolific septation following plo1+ overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1‐associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1‐associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36°C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1+ overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.

Synthesis of Empty Bacterial Microcompartments, Directed Organelle Protein Incorporation, and Evidence of Filament-Associated Organelle Movement

Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse structures can be generated by changing the expression profile of these genes, including the formation of large axial filaments that interfere with septation. Fusing GFP to PduC, PduD, or PduV, none of which are shell proteins, allows regiospecific targeting of the reporter group to the empty BMC. Live cell imaging provides unexpected evidence of filament-associated BMC movement within the cell in the presence of PduV.

Tropomyosin - master regulator of actin filament function in the cytoskeleton

ABSTRACT Tropomyosin (Tpm) isoforms are the master regulators of the functions of individual actin filaments in fungi and metazoans. Tpms are coiled-coil parallel dimers that form a head-to-tail polymer along the length of actin filaments. Yeast only has two Tpm isoforms, whereas mammals have over 40. Each cytoskeletal actin filament contains a homopolymer of Tpm homodimers, resulting in a filament of uniform Tpm composition along its length. Evidence for this ‘master regulator’ role is based on four core sets of observation. First, spatially and functionally distinct actin filaments contain different Tpm isoforms, and recent data suggest that members of the formin family of actin filament nucleators can specify which Tpm isoform is added to the growing actin filament. Second, Tpms regulate whole-organism physiology in terms of morphogenesis, cell proliferation, vesicle trafficking, biomechanics, glucose metabolism and organ size in an isoform-specific manner. Third, Tpms achieve these functional outputs by regulating the interaction of actin filaments with myosin motors and actin-binding proteins in an isoform-specific manner. Last, the assembly of complex structures, such as stress fibers and podosomes involves the collaboration of multiple types of actin filament specified by their Tpm composition. This allows the cell to specify actin filament function in time and space by simply specifying their Tpm isoform composition. Summary: This Commentary explains how tropomyosin isoforms determine the function of individual actin filaments in the cytoskeleton, and their subsequent use in directing cell structure and function.

Solution Structure of a Bacterial Microcompartment Targeting Peptide and Its Application in the Construction of an Ethanol Bioreactor

Targeting of proteins to bacterial microcompartments (BMCs) is mediated by an 18-amino-acid peptide sequence. Herein, we report the solution structure of the N-terminal targeting peptide (P18) of PduP, the aldehyde dehydrogenase associated with the 1,2-propanediol utilization metabolosome from Citrobacter freundii. The solution structure reveals the peptide to have a well-defined helical conformation along its whole length. Saturation transfer difference and transferred NOE NMR has highlighted the observed interaction surface on the peptide with its main interacting shell protein, PduK. By tagging both a pyruvate decarboxylase and an alcohol dehydrogenase with targeting peptides, it has been possible to direct these enzymes to empty BMCs in vivo and to generate an ethanol bioreactor. Not only are the purified, redesigned BMCs able to transform pyruvate into ethanol efficiently, but the strains containing the modified BMCs produce elevated levels of alcohol.

The recruitment of acetylated and unacetylated tropomyosin to distinct actin polymers permits the discrete regulation of specific myosins in fission yeast.

Tropomyosin (Tm) is a conserved dimeric coiled-coil protein, which forms polymers that curl around actin filaments in order to regulate actomyosin function. Acetylation of the Tm N-terminal methionine strengthens end-to-end bonds, which enhances actin binding as well as the ability of Tm to regulate myosin motor activity in both muscle and non-muscle cells. In this study we explore the function of each Tm form within fission yeast cells. Electron microscopy and live cell imaging revealed that acetylated and unacetylated Tm associate with distinct actin structures within the cell, and that each form has a profound effect upon the shape and integrity of the polymeric actin filament. We show that, whereas Tm acetylation is required to regulate the in vivo motility of class II myosins, acetylated Tm had no effect on the motility of class I and V myosins. These findings illustrate a novel Tm-acetylation-state-dependent mechanism for regulating specific actomyosin cytoskeletal interactions.

Targeted amino-terminal acetylation of recombinant proteins in E. coli.

One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

Formins determine the functional properties of actin filaments in yeast.

The actin cytoskeleton executes a broad range of essential functions within a living cell. The dynamic nature of the actin polymer is modulated to facilitate specific cellular processes at discrete locations by actin-binding proteins (ABPs), including the formins and tropomyosins (Tms). Formins nucleate actin polymers, while Tms are conserved dimeric proteins that form polymers along the length of actin filaments. Cells possess different Tm isoforms, each capable of differentially regulating the dynamic and functional properties of the actin polymer. However, the mechanism by which a particular Tm localizes to a specific actin polymer is unknown. Here we show that specific formin family members dictate which Tm isoform will associate with a particular actin filament to modulate its dynamic and functional properties at specific cellular locations. Exchanging the localization of the fission yeast formins For3 and Cdc12 results in an exchange in localizations of Tm forms on actin polymers. This nucleator-driven switch in filament composition is reflected in a switch in actin dynamics, together with a corresponding change in the filaments ability to regulate ABPs and myosin motor activity. These data establish a role for formins in dictating which specific Tm variant will associate with a growing actin filament and therefore specify the functional capacity of the actin filaments that they create.

Cytokinetic actomyosin ring formation and septation in fission yeast are dependent on the full recruitment of the polo-like kinase Plo1 to the spindle pole body and a functional spindle assembly checkpoint.

In dividing cells, the assembly and contraction of the cytokinetic actomyosin ring (CAR) is precisely coordinated with spindle formation and chromosome segregation. Despite having a cell wall, the fission yeast Schizosaccharomyces pombe forms a CAR reminiscent of the structure responsible for the cleavage of cells with flexible boundaries. We used the myo2-gc fission yeast strain in which the chromosomal copy of the type II myosin gene, myo2+, is fused to the gene encoding green fluorescent protein (GFP) to investigate the dynamics of Myo2 recruitment to the cytokinetic actomyosin ring in living cells. Analysis of CAR formation in relation to spindle pole body (SPB) and centromere separation enabled us to pinpoint the timing of Myo2 recruitment into a stable CAR structure to the onset of anaphase A. Depolymerisation of actin with latrunculin B did not affect the timing of Myo2 accumulation at the cell equator (although Myo2 no longer formed a ring), whereas depolymerisation of microtubules with either thiabendazole (TBZ) or methyl 2-benzimidazolecarbamate (MBC) resulted in a delay of up to 90 minutes in CAR formation. Microtubule depolymerisation also delayed the localisation of other CAR components such as actin and Mid1/Dmf1. The delay of cytokinesis in response to loss of microtubule integrity was abolished in cells lacking the spindle assembly checkpoint protein Mad2 or containing non-functional Cdc16, a component of the fission yeast septation initiation network (SIN). The delay was also abolished in cells lacking Zfs1, a component of the previously described S. pombe cytokinesis checkpoint. Recruitment of the polo-related kinase, Plo1, a key regulator of CAR formation, to the SPBs was substantially reduced in TBZ in a Mad2-dependent manner. Loading of Cdc7, a component of the SIN and downstream of Plo1 in the cytokinesis pathway, onto the the SPBs was also delayed in TBZ to the same extent as CAR formation. We conclude that CAR formation is subject to regulation by the spindle assembly checkpoint via the loading of Plo1 onto the SPBs and the consequent activation of the SIN.

Myosin V-mediated vacuole distribution and fusion in fission yeast

The class V myosins are actin-based motors that move a variety of cellular cargoes [1]. In budding yeast, their activity includes the relocation of a portion of the vacuole from the mother cell to the bud [2, 3]. Fission yeast cells contain numerous (approximately 80) small vacuoles. When S. pombe cells are placed in water, vacuoles fuse in response to osmotic stress [4]. Fission yeast possess two type V myosin genes, myo51(+) and myo52(+) [5]. In a myo51Delta strain, vacuoles were distributed throughout the cell, and mean vacuole diameter was identical to that seen in wild-type cells. When myo51Delta and wild-type cells were placed in water, vacuoles enlarged by fusion. In myo52Delta cells, by contrast, vacuoles were smaller and mostly clustered around the nucleus, and fusion in water was largely inhibited. When cells containing GFP-Myo52 were placed in water, Myo52 was seen to redistribute from the cell poles to the surface of the fusing vacuoles. Vacuole fusion in fission yeast was inhibited by the microtubule drug thiabendazole (TBZ) but not by the actin inhibitor latrunculin B. This is the first demonstration of the involvement of a type V myosin, possibly via an interaction with microtubules, in homotypic membrane fusion.

In vivo movement of the type V myosin Myo52 requires dimerisation but is independent of the neck domain

Intracellular movement is a fundamental property of all cell types. Many organelles and molecules are actively transported throughout the cytoplasm by molecular motors, such as the dimeric type V myosins. These possess a long neck, which contains an IQ motif, that allow it to make 36-nm steps along the actin polymer. Live cell imaging of the fission yeast type V myosin Myo52 reveals that the protein moves rapidly throughout the cytoplasm. Here, we describe analysis of this movement and have established that Myo52 moves long distances on actin filaments in an ATP-dependent manner at ∼0.5 μm/second. Myo51 and the microtubule cytoskeleton have no discernable role in modulating Myo52 movements, whereas rigour mutations in Myo52 abrogated its movement. We go on to show that, although dimerisation is required for Myo52 movement, deleting its neck has no discernable affect on Myo52 function or velocity in vivo.