Figure 3. Naegleria’s spindle is a barrel shape composed of bundles of microtubules that elongate as mitosis proceeds.

(A) Asynchronously growing Naegleria amoebae were fixed and stained with anti-α-tubulin clone DM1A (green) to detect microtubules and DAPI to label DNA (magenta). Mitotic spindles were imaged using confocal microscopy (top row), and images were deconvolved using Autoquant software (bottom rows). Cells were classified as prophase, metaphase, or anaphase/telophase.

(B) Quantification of maximum spindle length (left) and the spindle width at half the length (right). Each point represents one mitotic spindle, and lines indicate the averages (from 3 experimental replicates encompassing the following numbers of cells: prophase, n = 6; metaphase, n = 10; anaphase/telophase, n = 4). Spindles were imaged and deconvolved as in (A).

(C) Orthogonal views of a metaphase spindle (imaged and deconvolved as in A) lying in the plane of the coverslip; XZ and YZ views generated in Fiji.

(D) Structured illumination microscopy of a spindle lying perpendicular to the coverslip.

(E) Confocal microscopy and deconvolution of nucleoli in mitotic Naegleria. Cells were fixed and stained to detect tubulin (YOL 1/34 antibody, green, top panels only), DNA (DAPI, magenta), and nucleolar protein (DE6 antibody, cyan). One maximum intensity projection is shown (top cell), while remaining images are single z planes.

(F) Transmission electron microscopy of microtubule bundles in Naegleria; arrowheads indicate microtubule bundles and boxed regions (left) are shown as enlarged insets (right).

See also Figures S3–S5 and Table S2.