Fig 2: Airway cells in urethane-induced lung tumors.(A) Cartoon of the different lung epithelial lineages, their distribution in the airways (club, goblet, ciliated, and basal cells) and the alveoli (alveolar type I and II cells), their permanent fluorescent genetic labeling in the reporter mice used in this study (green color), and the protein markers used for their identification. See also Figure 1—figure supplements 1–5. (B) Lung sections from naïve 6-week-old GFP;CCSP.CRE mice (n = 22), in which all airway cells bear permanent genetic GFP+ (green arrows) and all other cells TOMATO+ (red arrows) labels, counterstained with nuclear Hoechst33258 dye (top) or immunostained for the club cell marker CCSP and the alveolar type II cell marker SFTPC (bottom). a, alveoli; b, bronchi; v, vein. See also Figure 1—figure supplements 6–8. (C) Proliferating cell nuclear antigen (PCNA; brown) and hematoxylin (blue)-stained (top) and CCSP (green) and Hoechst33258 (blue)-stained (bottom) lung tumor sections of urethane-treated C57BL/6 mice six months post-treatment (n = 5/group), depicting endobronchial lung adenocarcinomas (white arrows). See also Figure 1—figure supplements 9–11. (D) Lung sections of GFP;CCSP.CRE mice (n = 10) at six months post-urethane treatment bearing hyperplasias and tumors (dashed outlines, top), and immunostained for the club cell marker CCSP (bottom left) and the alveolar type II cell marker SFTPC (bottom right). Note the GFP-labeled lesions of airway origin that have lost CCSP and have acquired SFTPC immunoreactivity. See also Figure 1—figure supplements 12–19. CCSP, Clara cell secretory protein; TUBA1A, acetylated a-tubulin; SFTPC, surfactant protein C; LYZ2, lysozyme 2; FOXJ1, forkhead box J1; KRT5, keratin 5.

Fig 3: TUBA1A-V409I/A mutants alter neuron morphologies.(A) Representative days in vitro 2 (DIV2) cortical neurons expressing cytoplasmic GFP and 6X-His-tagged TUBA1A-WT, -V409I, and -V409A. Scale bar = 10 µm. (B) Quantification of number of primary, secondary, and tertiary branches along axons and dendrites in each condition. Images were collected from three independent experiments; n=65 cells for wild-type (WT), n=56 for V409I, and n=59 for V409A. Each dot represents a single cell. Bars are mean ± 95% confidence interval. Statistical analysis between multiple groups was analyzed by two-way ANOVA and corrected for multiple comparisons post hoc by Tukey test. All statistics with p=0.05 are indicated on graph. (C) Example image of DIV2 cortical neuron expressing cytoplasmic GFP and TUBA1A-WT. Scale bar = 10 µm. Yellow box indicates a representative region measured every 30 s for 15 min at 37°C, 5% CO2. Scale bar in insets = 5 µm. (D) Proportion of events classified as the neurite growing, paused, or shrinking, depending on if the length of the neurite grew, stayed the same, or shrunk, respectively. Images were collected from three independent experiments; n=10 cells for WT, n=13 for V409I, and n=13 for V409A. Bars are mean ± standard error of the mean. (E) Quantification of the amount of time a neurite was in a paused state, neither growing nor shrinking. Each dot represents a period of pause; cell numbers are the same as stated in (D). Bars are mean ± 95% confidence interval. Statistical analysis was done using an unpaired t-test. All statistics with p=0.05 are indicated on graph. (F) Representative images of neurons expressing the above plasmids, exposed to 4°C for indicated time, and stained with TUBB2A/B. Scale bar = 10 µm. (G) Quantification of the number of neurites per cell every 2 hr over a 6 hr 4°C cold shock. Images were collected from three independent experiments; for WT n=36 cells at 0 hr, n=35 at 2 hr, n=41 at 4 hr, n=44 at 6 hr; for V409I n=32 at 0 hr, n=30 at 2 hr, n=27 at 4 hr, n=29 at 6 hr; for V409A n=37 at 0 hr, n=29 at 2 hr, n=27 at 4 hr, n=22 at 6 hr. Dots represent averages from the three separate experiments and error bars are ± 95% confidence interval. (H) The proportion of neurites that have retraction bulbs per cell measured every 2 hr over a 6 hr 4°C cold shock. Cell numbers and error bars are the same as stated in (G). Figure 2—source data 1.Number of processes per cell. Figure 2—source data 2.Neurite dynamics under standard conditions. Figure 2—source data 3.Number of neurites and retraction bulbs under cold temperature conditions.

Fig 4: Advantages of using small probes in imaging. (A) 2D STED image of a late cytokinetic furrow in cells labelled using an antibody to acetylated tubulin (T7451, Sigma-Aldrich, shown in magenta; signal is from a combination of primary and secondary antibodies) and the tubulin Affimer (Affimer 32, shown in green). These images, summarised in the diagram below, show how densely packed microtubules (25 nm wide) in the central region of the midbody (so-called ‘cut-zone’, arrowed in the STED images) are labelled by the smaller Affimer probe (less than 4 nm long), but not by the more commonly used combination of primary and secondary antibodies (each ~15 nm long) (see also Tiede et al., 2017). (B) 2D STED image of Z-discs in cardiac muscle tissue, labelled with an antibody to the Z-disc protein ACTN2 (A7732, Sigma-Aldrich; ACTN2) using a combination of primary and secondary antibodies (magenta), and an Affimer to ACTN2 (green). The diagram below illustrates the structures of F-actin, full-length ACTN2 (PDB 4D1E), the ACTN2 Affimer bound to the calponin homology (CH) domains of ACTN2 (PDB 6SWT) (Curd et al., 2021) and primary and secondary antibodies (PDB 1IGY) drawn to scale, for part of the Z-disc. The anti-parallel ACTN2 dimer crosslinks actin filaments (derived from sarcomeres on either side). The combination of primary and secondary antibodies (>20 nm in length) is less able to penetrate the full width of the Z-disc (see arrows on STED images) as illustrated here, accounting for reduced ACTN2 staining in the central region of the Z-disc. The Affimer is much smaller and able to penetrate the entire Z-disc. The restricted localisation of antibodies is noticeable in STED, with a resolution of ~50 nm, but would not be seen in confocal microscopy, with a resolution of ~250 nm.

Fig 5: Mechanisms of Tuba1a-induced axonal pathfinding deficits. (A) Schematic illustrating how reduced Tuba1a may impair ability of Tuba1a ND/+ axons to reach key signaling points at the correct developmental time to support proper brain formation. Wild-type axons (WT; left) reaching the midline crossing point of corpus callosum by embryonic day (E) 18.5 are compared to the potentially stunted axonal growth in Tuba1a ND/+ brains (right). The reduced density of microtubules impair localization of Map1b and potentially other MAPs to contribute to slower growth of developing Tuba1a ND/+ commissural neurons.