Septin filaments exhibit a dynamic, paired organization that is conserved from yeast to mammals

Abstract

The septins are conserved, GTP-binding proteins important for cytokinesis, membrane compartmentalization, and exocytosis. However, it is unknown how septins are arranged within higher-order structures in cells. To determine the organization of septins in live cells, we developed a polarized fluorescence microscopy system to monitor the orientation of GFP dipole moments with high spatial and temporal resolution. When GFP was fused to septins, the arrangement of GFP dipoles reflected the underlying septin organization. We demonstrated in a filamentous fungus, a budding yeast, and a mammalian epithelial cell line that septin proteins were organized in an identical highly ordered fashion. Fluorescence anisotropy measurements indicated that septin filaments organized into pairs within live cells, just as has been observed in vitro. Additional support for the formation of pairs came from the observation of paired filaments at the cortex of cells using electron microscopy. Furthermore, we found that highly ordered septin structures exchanged subunits and rapidly rearranged. We conclude that septins assemble into dynamic, paired filaments in vivo and that this organization is conserved from yeast to mammals.

Figure 1.

Analysis of constrained septin–GFP fusions using polarized fluorescence microscopy shows that septin rings in A. gossypii are ordered. (A) Raw fluorescence acquired by exciting GFPs in an A. gossypii septin ring with four angles of polarized light in a cell expressing Cdc12-conGFP4. Cell outline is shown in white. Bar, 1 µm. (B) Maps of polarization ratio (pr) and GFP dipole angle (azimuth) were calculated for every camera pixel based on the data in A. The pr across the ring is expressed with the angle as blue lines of a length proportional to the pr, oriented according to the calculated azimuth and overlayed on the sum fluorescence (“pr-scaled” image). Alternatively, the amount and orientation of ordered protein is represented using a color scheme (“spectrum” look-up table in ImageJ) in which each angle is represented as a color whose intensity is the product of the fluorescence and the pr (“pr*fluor-scaled angles” image). Bar, 1 µm. (C) Representation of the “offset” angle (θ), which is the distance counterclockwise from the cell growth axis to the mean azimuth. Two perspectives of an A. gossypii septin ring viewed in the xy or top view (left ring) and the xz or cross section view (right ring).

Figure 2.

Septin rings and multiple septin subunits are ordered throughout A. gossypii development. (A) A. gossypii septin rings analyzed at their top, middle, and bottom planes were measured to have GFP dipoles oriented perpendicular (or parallel, depending on the strain imaged) to the cell growth axis. The same septin ring shown in Fig. 1 (A and B), from a cell expressing Cdc12-conGFP4, is pictured here at different focal planes. The amount and azimuth of ordered septin protein is shown using a color scale (“pr*fluor-scaled angles” image). Cells expressing Cdc12-conGFP4 or Cdc3-conGFP from a replicating plasmid were imaged using polarized excitation and analyzed. Fluorescence (left), fluorescence overlayed with blue lines scaled in length by pr and oriented at the calculated GFP dipole angle (azimuth) for each camera pixel (middle), and the amount and azimuth of ordered septin protein using a color scale (right) are displayed. (B) Septins before ring assembly are ordered at growing hyphal tips. The typical azimuth orientation is perpendicular to the cell cortex. (C) Septin rings formed at branch points are highly ordered. The typical azimuth orientation is perpendicular to the cell growth axis. (D) An inter-region (IR) septin ring, assembled in the wake of a growing tip in a cell expressing Cdc3-conGFP. Septin rings in cells expressing this Cdc3-conGFP construct exhibit similar order, with the typical azimuth orientation perpendicular to the cell growth axis. Cell outlines are shown in white. Bars, 1 µm.

Figure 3.

Ordered septin filaments are paired, align parallel to the cell growth axis, and are dynamic. (A) A. gossypii cells expressing Cdc12-conGFP4 were treated with the septin filament stabilizing drug FCF. The mean GFP dipole orientation in septin fibers was found to be perpendicular to the fiber axis. Cell outlines are shown in white. Bar, 1 µm. (B) A. gossypii cells (AG127) were treated for 3 h with FCF and processed for TEM. A typical filament and a magnified section are displayed. Brackets point to the measured width and periodicity of a population of septin filaments gathered from many different sections accompanied by standard deviations (statistics are given in Table S1). PM, plasma membrane; CW, cell wall. (C) An immunolabeled section of SHS1-6HA–expressing cells (AG296) in which septins have been localized using an anti-HA primary and a 10-nm gold conjugated secondary antibody. Arrowheads point to gold particles. Bar, 100 nm. (D) A. gossypii cells expressing Cdc12-conGFP4 or Cdc3-conGFP exhibit GFP dipoles oriented parallel to the cell cortex when imaged in cross section (xz plane). Cell outlines are shown in white. Bars, 1 µm. (E) A septin ring in A. gossypii expressing Cdc12-conGFP4 was analyzed, photobleached and analyzed, and allowed to recover before repeat of analysis. The recovered signal is ordered and reports the same GFP dipole orientation (perpendicular to the cell growth axis) as the starting ring. Bar, 1 µm.

Figure 4.

Septin rings in S. cerevisiae and A. gossypii are similarly ordered and accomplish an ∼90° change in orientation without concerted rotation. (A) Septin rings assembled in S. cerevisiae and A. gossypii expressing Cdc12-conGFP4 exhibit septins organized such that, on average, GFP dipoles are oriented perpendicular to the cell growth axis. As in A. gossypii (Fig. 3C), the S. cerevisiae septin hourglass, when viewed in cross section, also has the average GFP dipole position oriented parallel to the cell cortex, and perpendicular to the growth axis. Cell outlines are shown in white. Bars, 1 µm. (B) Split septin rings show an ∼90° change in the orientation of the GFP dipoles. Some septin rings in A. gossypii expressing Cdc12-conGFP4 were captured in a partially split state, exhibiting the original (perpendicular) and reorganized (parallel) orientation of dipoles. (C) Septin rings in S. cerevisiae expressing Cdc12-conGFP4 were imaged using every minute, through the transition from the hourglass to a split ring. (D) Polarization ratio of the central region (12 pixels) of S. cerevisiae septin rings as they progressed through the septin reorganization were analyzed and plotted. Average orientations perpendicular to the ring axis are plotted as negative values and average orientations parallel to the mother–bud axis are plotted as positive values to represent the difference in orientation seen through time. n = 3; error bars are standard deviation.

Figure 5.

Mammalian epithelial cells exhibit conserved septin order. (A) MDCK cells expressing SEPT2–conGFP-1-344, which incorporates into septin fibers. The GFP dipoles are measured to be oriented perpendicular to the fiber axis. The white box identifies the magnified region shown in B. Bar, 10 µm. (B) Magnified region of A including panels with scaled azimuth lines and color-intensity schemes. Bar, 1 µm.

Figure 6.

Septin filament organization models and corresponding changes in dipole orientation. (A) Representations of a single septin protofilament shown associated with the cortex (blue) with the conGFP-tagged septin (green) shown with a possible GFP dipole orientation (black). C-terminal coiled-coil domains are shown as rectangles extending from the three septins that are predicted to have these domains. The left and right half of a filament are related by a 180° rotation around a vertical axis shown in the side view as a dotted line and red arrow. The summed angle for all possible dipole orientations is noted on the right. (B) Representations of paired septin filaments. Top and bottom filaments are related by a 180° rotation around a horizontal axis. The summed angle for all possible dipole orientations is noted on the right. (C) Paired filament models show polymerization of filament if it processes straight or twisted relative to the cortex. Corresponding dipole angles of tagged subunits are represented by the arrows. (D) The same filament procession and relative dipole angles that could be captured in the axial resolution of a 1.4 NA objective (∼500 nm) when viewed in cross section. The dipole orientations for each organizational scenario is shown below the filament and noted for anisotropy. (E) Model for filament organization in the S. cerevisiae hourglass (left), transition (middle), and split ring (right) states that is consistent with anisotropy measurements through time.