An alternate route for cellulose microfibril biosynthesis in plants.

Similar to cellulose synthases (CESAs), cellulose synthase-like D (CSLD) proteins synthesize β-1,4-glucan in plants. CSLDs are important for tip growth and cytokinesis, but it was unknown whether they form membrane complexes in vivo or produce microfibrillar cellulose. We produced viable CESA-deficient mutants of the moss to investigate CSLD function without interfering CESA activity.

Cellulose synthase-like D movement in the plasma membrane requires enzymatic activity.

Cellulose Synthase-Like D (CSLD) proteins, important for tip growth and cell division, are known to generate β-1,4-glucan. However, whether they are propelled in the membrane as the glucan chains they produce assemble into microfibrils is unknown. To address this, we endogenously tagged all eight CSLDs in Physcomitrium patens and discovered that they all localize to the apex of tip-growing cells and to the cell plate during cytokinesis.

CRISPR-Cas9 Genome Editing in the Moss Physcomitrium (Formerly Physcomitrella) patens.

Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR-Cas9 mediated genome editing, P.

Slip slidin' away: Bristle-driven gliding by Tetradesmus deserticola (Chlorophyta) in microfluidic chambers.

Microalgae within the Scenedesmaceae are often distinguished by spines, bristles, and other wall characteristics. We examined the dynamic production and chemical nature of bristles extruded from the poles of Tetradesmus deserticola previously isolated from microbiotic crust. Rapidly growing cells in a liquid growth medium were established in polydimethylsiloxane microfluidic chambers specially designed to maintain aerobic conditions over time within a chamber 6-12 μm deep.

COPII Sec23 proteins form isoform-specific endoplasmic reticulum exit sites with differential effects on polarized growth.

Coat Protein complex II (COPII), a coat protein complex that forms vesicles on the endoplasmic reticulum (ER), mediates trafficking to the Golgi. While metazoans have few genes encoding each COPII component, plants have expanded these gene families, leading to the hypothesis that plant COPII has functionally diversified. In the moss Physcomitrium (Physcomitrella) patens, the Sec23/24 gene families are each composed of seven genes.

The Moss () : A Model Organism for Non-Seed Plants.

Since the discovery two decades ago that transgenes are efficiently integrated into the genome of by homologous recombination, this moss has been a premier model system to study evolutionary developmental biology questions, stem cell reprogramming, and the biology of nonvascular plants. was the first non-seed plant to have its genome sequenced. With this level of genomic information, together with increasing molecular genetic tools, a large number of reverse genetic studies have propelled the use of this model system.

analysis of formin dynamics in the moss reveals functional class diversification.

Formins are actin regulators critical for diverse processes across eukaryotes. With many formins in plants and animals, it has been challenging to determine formin function We found that the phylogenetically distinct class I integral membrane formins (denoted For1) from the moss enrich at sites of membrane turnover, with For1D more tightly associated with the plasma membrane than For1A. To probe formin function, we generated formin-null lines with greatly reduced formin complexity.

Cytoskeletal discoveries in the plant lineage using the moss Physcomitrella patens.

Advances in cell biology have been largely driven by pioneering work in model systems, the majority of which are from one major eukaryotic lineage, the opisthokonts. However, with the explosion of genomic information in many lineages, it has become clear that eukaryotes have incredible diversity in many cellular systems, including the cytoskeleton. By identifying model systems in diverse lineages, it may be possible to begin to understand the evolutionary origins of the eukaryotic cytoskeleton.

Actin and microtubule cross talk mediates persistent polarized growth.

Coordination between actin and microtubules is important for numerous cellular processes in diverse eukaryotes. In plants, tip-growing cells require actin for cell expansion and microtubules for orientation of cell expansion, but how the two cytoskeletons are linked is an open question. In tip-growing cells of the moss , we show that an actin cluster near the cell apex dictates the direction of rapid cell expansion.

An ancient Sec10-formin fusion provides insights into actin-mediated regulation of exocytosis.

Exocytosis, facilitated by the exocyst, is fundamentally important for remodeling cell walls and membranes. Here, we analyzed , a novel gene that encodes a fusion of an exocyst subunit (Sec10) and an actin nucleation factor (formin). We showed that the fusion occurred early in moss evolution and has been retained for more than 170 million years.

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane.

Long-Term Growth of Moss in Microfluidic Devices Enables Subcellular Studies in Development.

Key developmental processes that occur on the subcellular and cellular level or occur in occluded tissues are difficult to access, let alone image and analyze. Recently, culturing living samples within polydimethylsiloxane (PDMS) microfluidic devices has facilitated the study of hard-to-reach developmental events. Here, we show that an early diverging land plant, Physcomitrella patens, can be continuously cultured within PDMS microfluidic chambers.

Myosin VIII associates with microtubule ends and together with actin plays a role in guiding plant cell division.

Plant cells divide using the phragmoplast, a microtubule-based structure that directs vesicles secretion to the nascent cell plate. The phragmoplast forms at the cell center and expands to reach a specified site at the cell periphery, tens or hundreds of microns distant. The mechanism responsible for guiding the phragmoplast remains largely unknown.

Transient RNAi assay in 96-well plate format facilitates high-throughput gene function studies in planta.

Here we describe a rapid high-throughput method for performing RNA interference (RNAi) in moss, in which phenotyping is performed within 1 week after transformation. The moss Physcomitrella patens is a great plant model system for reverse genetic studies due to its amenability to homologous recombination as well as RNAi. Our lab has developed a rapid RNAi assay to screen for growth phenotypes in moss protonemal tissue.

Class II formin targeting to the cell cortex by binding PI(3,5)P(2) is essential for polarized growth.

Class II formins are key regulators of actin and are essential for polarized plant cell growth. Here, we show that the class II formin N-terminal phosphatase and tensin (PTEN) domain binds phosphoinositide-3,5-bisphosphate (PI(3,5)P(2)). Replacing the PTEN domain with polypeptides of known lipid-binding specificity, we show that PI(3,5)P(2) binding was required for formin-mediated polarized growth.

Myosin VIII regulates protonemal patterning and developmental timing in the moss Physcomitrella patens.

Plants have two classes of myosins. While recent work has focused on class XI myosins showing that myosin XI is responsible for organelle motility and cytoplasmic streaming, much less is known about the role of myosin VIII in plant growth and development. We have used a combination of RNAi and insertional knockouts to probe myosin VIII function in the moss Physcomitrella patens.

Dimerization is important for the GTPase activity of chloroplast translocon components atToc33 and psToc159.

Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg(130)) has been implicated in the formation of the atToc33 dimer and in intermolecular GTPase activation within the dimer. Here we report the crystal structure at 3.