The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape

Die Regulation der Zellpolarität in der Bäckerhefe <i>Saccharomyces cerevisiae</i>

Die Polarisierung der Saccharomyces cerevisiae-Zellen erfolgt zu verschiedenen Zeitpunkten während der Zellteilung. Zu Beginn der Zellteilung werden spezifische Orte für die Sprossung ausgewählt. Die Wahl der Sprossungsorte folgt definierten räumlichen Mustern und unterliegt der Kontrolle von Zelltyp und Nährstoffbedingungen. Diploide Hefezellen folgen bei der Sprosswahl einem bipolaren Muster, bei dem die Zellteilung mit gleicher Wahrscheinlichkeit entweder am Geburtsenden, dem proximalen Zellpol, oder am direkt gegenüber liegendem Ort, dem distalen Zellpol beginnt. Bei Stickstoffmangel wechseln diploide Zellen zu einem unipolar-distalen Sprossungsmuster. In dieser Arbeit wurden die Funktionen von zwei Zellpolproteinen, Bud8p und Bud9p, im Detail analysiert. Genetische und zellbiologische Analysen zeigten, dass Bud8p am distalen Zellpol lokalisiert ist und für die Sprossung am distalen Pol benötigt wird. Bud9p protein ist an beiden Zellpolen lokalisiert, wird aber nur für die proximale Sprossung gebraucht. Biochemische Analysen zeigten, dass Bud8p und Bud9p miteinander physikalisch interagieren. Bud8p fungiert deshalb vermutlich als Markerprotein für die distale Sprosswahl und wird dabei durch die Anwesenheit von Bud9p negativ beeinflusst. Dagegen Bud9p dient als Markerprotein für die Wahl der Sprossung am proximalen Zellpol. In weiteren Untersuchungen wurde die Funktion von Regulatoren des Aktinzytoskeletts (Typ I Myosine) bei der Sprosswahl analysiert. Genetische Analysen zeigten, dass Typ I Myosine Myo3p und Myo5p für die Erkennung der zellpole benötigt wird. Zusätzlich bindet Bud8p an Myo3p, was darauf hindeutet, dass Typ I Myosine durch Interaktion mit Markerproteinen die Sprosswahl durch ortspezifische Aktinpolymerisierung steuern könnte. Mittels Hefe-Two-Hybrid-System wurden neue Interaktionspartner von Bud8p isoliert und über biochemische Analysen weiter charakterisiert. Dabei wurden einige Proteine identifiziert, die an der Translation oder am zellulären Transport beteiligt sind, z. B. das ribosomale Protein Rpl12Ap, das Polysomen-assozierte und mRNA-bindende Protein Scp160p, sowie das am Vesikeltransport beteiligte Protein Trs120p. Diese Daten lassen vermuten, dass Bud8p Protein die Sprosswahl durch Beeinflussung des zellulären Transports und der Translation regulieren könnte

A simplified model for MyoE function at the hyphal tip.

<p>A. A <i>myoE</i>⁺ cell. Exocytic vesicles move along microtubules powered by kinesin molecules. (It is likely that several kinesins can carry out this function.) There is a large zone of overlap between microtubules and actin microfilaments. When exocytic vesicles become detached from microtubules, as will generally be the case because of the limited processivity of kinesins, MyoE, on the vesicles will move the vesicles along actin microfilaments, collecting them at the Spitzenkörper. The vesicles then fuse in a fairly small area to the plasma membrane releasing their contents and resulting in hyphal growth. MyoE, vesicle components and, probably, many more proteins are moved in retrograde direction by dynein where they will be reused. B. A <i>myoE</i>Δ cell. In the absence of MyoE, exocytic vesicles are not focused into the Spitzenkörper but they are still moved into the hyphal apex area where they fuse with the plasma membrane over a wider area, resulting in hyphae with a greater diameter and lower extension rate. For simplicity, much of the endocytic machinery including endosomes and actin patches has been left out of this model. For a more detailed model of the endocytic machinery please see reference 10.</p

MyoE localization.

<p>A–F are images of the same field and are maximum intensity projections of a Z-series stack. A–C show the co-localization of MyoE-GFP and mCherry-SynA at the Spitzenkörper (arrows). SynA localizes to the Spitzenkörper and to the plasma membrane near the apex (B). In D–F, the thresholds are chosen to reveal the punctate staining in the hypha while overexposing the MyoE-GFP and mCherry-SynA at the hyphal tip. MyoE-GFP localizes to numerous small puncta and some larger structures that may be endosomes (e.g. arrow). G. Faint localization of MyoE-GFP at forming septa (arrows). H. A three-dimensional projection of a hyphal tip showing MyoE-GFP and mCherry-SynA. Although MyoE and SynA co-localize at the Spitzenkörper, many puncta behind the tip show GFP fluorescence or mCherry fluorescence, but it was not clear that there was any obligate co-localization.</p

Movement of MyoE-GFP to the Spitzenkörper in the absence of microtubules.

<p>Microtubules have been depolymerized with 2.4 µg/ml benomyl time at the upper right in each panel is in seconds. At t = 0, MyoE-GPF is visible at the Spitzenkörper (arrow). Three seconds later after FRAP the MyoE-GFP in the Spitzenkörper is bleached. In spite of the absence of microtubules, MyoE-GFP has moved to the Spitzenkörper 30 sec after FRAP (arrow, t = 33) and it increases in intensity at the Spitzenkörper over the next minute (arrows).</p

Deletion of myoB inhibits septum formation.

<p>All panels are images of living cells. In A and B, nuclei are shown with histone H1-mRFP and chitin is stained with calcofluor (10 µg/ml). A. a <i>myoB</i>⁺ strain (LO1516). Multiple septa are visible (arrows). B. a <i>myoB</i>Δ hypha. The <i>myoB</i> gene was deleted in LO1516 and nuclei carrying the deletion were maintained in a heterokaryon. No septa are present but there are thickened regions containing chitin (e.g. arrow) and chitin is highly concentrated near the hyphal tip. C. Shows a hyhal tip region in a <i>myoB</i>Δ strain stained with calcofluor but nuclei are not imaged. Note the absence of septa and side branches. The circular objects are ungerminated conidia resulting from the heterokaryon rescue technique.</p

Deletion of myoE alters hyphal morphology and SynA distribution but not the localization of endocytic patches.

<p>Panel A shows a <i>myoE</i>⁺ strain and panel B shows a <i>myoE</i>Δ strain. Both are stained with 10 µg/ml calcofluor. Hyphae in the <i>myoE</i>Δ strain are thicker, vary more in thickness and exhibit more branching near the tip. The amount of chitin staining at the hyphal tip varied from hypha to hypha in wild-type strains as well as <i>myoB</i> and <i>myoE</i> deletion strains. The difference in staining between A and B is not specific to myoEΔ. Panel C shows GFP-SynA in a <i>myoE</i>⁺ strain. SynA is concentrated into the Spitzenkörper at the hyphal tip (arrow) and is also present at the membrane near the tip. Panel D shows GFP-SynA in a <i>myoE</i>Δ strain. SynA is present at the membrane and in puncta in the cytoplasm but is not obviously organized into a Spitzenkörper. Panel E shows the localization of AbpA-mRFP and GFP-SynA in a <i>myoE</i>Δ strain. The image is a single focal plane from a deconvolved Z-series stack. AbpA-containing endocytic patches (arrow) localize to the cortex behind the growing tip and in three dimensions form a collar behind the growing tip. Panel F shows a control <i>myoE</i>⁺ strain (LO1548) also expressing GFP-SynA and AbpA-mRFP. The image is a single focal plane from a deconvolved Z-series stack. The ApbA-containing patches (arrow) appear to be organized into a tighter array and the Spitzenkörper is visible (arrowhead). Note that <i>myoE</i>⁺ hyphae are more consistent in diameter along their length than <i>myoE</i>Δ hyphae (compare A and B) and that the apices in <i>myoE</i>Δ hyphae appear rounder than in <i>myoE</i>⁺ hyphae. A and B are the same magnification as are C and D. Panel G shows branching ahead of the first septum (septum designated with an arrow).</p

Growth phenotype of myosin deletants.

<p>Incubation was for three days at 37°C on YAG medium supplemented with riboflavin. While both <i>myoB</i> and <i>myoE</i> deletants are viable, the <i>myoB</i> deletant colony is thin and wispy. Microscopic examination revealed that individual hyphae extend beyond the apparent edge of the colony. The <i>myoE</i> deletant is compact, exhibiting slower radial growth than the control strain.</p

Cytochalasin A causes MyoE-GFP to disperse from the Spitzenkörper.

<p>Images are maximum intensity projections of Z-series stacks. Time (in sec) after the addition of DMSO (top row) or an equivalent volume of cytochalasin A dissolved in DMSO to give a final concentration of 1 µg/ml (bottom row). MyoE-GFP continuously localizes to the Spitzenkörper in the solvent control (top row) but disperses in less than 328 sec after the addition of cytochalasin A.</p

Fluorescence recovery after photobleaching (FRAP) of GFP-SynA in myoE+ and myoEΔ strains.

<p>The tips of the two strains were photobleached at T = 0 (sec). In the <i>myoE</i>+ strain recovery is rapid. GFP-SynA appears at the tip within 30 sec of photobleaching and quickly localizes to the Spitzenkörper (arrows). This indicates that vesicles with SynA move rapidly to the tip and move through the Spitzenkörper before fusing with the plasma membrane. In the <i>myoE</i>Δ strain, the GFP-SynA is also visible at the tip at 30 sec after bleaching. MYOE, thus is not required for movement of GFP-SynA-containing vesicles to the tip. The GFP-SynA does not go through the Spitzenkörper, moreover, but fuses with the plasma membrane in a broad region of the tip.</p