Sir actin

Cells grown on 12-mm No. 1.5 coverslips (Carolina Biological Supply) were fixed with 4% paraformaldehyde (Electron Microscopy Sciences), washed with PBS, and permeabilized with either 0.1% TX100 or 0.1% saponin in a 3% BSA/PBS buffer. Subsequent primary and secondary antibody incubations were carried out in the permeabilization buffer. Coverslips were mounted in ProLong Gold reagent supplemented with DAPI (Invitrogen). Images were acquired with either a Zeiss LSM 800 laser scanning confocal microscope with a 63× Plan Apo (NA = 1.4) oil immersion objective and Zeiss Efficient Navigation software or an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer) that consisted of a Nikon Ti-E Eclipse inverted microscope equipped with 60× CFI PlanApo VC, NA 1.4, oil immersion objective and a CSU-X1 (Yokogawa) scan head that was driven by Volocity (PerkinElmer) software. For live cell imaging analysis of microtubules and F-actin, the cells were labeled with SiR-tubulin and SiR-actin (Cytoskeleton Inc.) as per the manufacturer’s instructions. An automated analysis pipeline was developed with Cell Profiler (McQuin et al, 2018 (link)) for the quantification of nuclear versus cytoplasmic ratios of the NLS-td-Tomato-NES reporter (Zhang et al, 2015 (link)).

Live cell imaging was performed on cumulus-enclosed oocytes recovered from mated females. Cumulus-enclosed oocytes were treated with hyaluronidase (30IU/ml) for 10 min to remove cumulus cells, then incubated in 500ul of KSOM-BSA for 30 min at 37°C and 5% CO2 in a humidified incubator. Then 0.5 µl of SiR-actin (Cytoskeleton Inc. Denver, CO) (1 mM) was added to achieve a final concentration of 1 µM and the oocytes were returned to the incubator for an additional 30 min. Oocytes were then washed in KSOM-BSA and transferred to a poly A-lysine (Sigma-Aldrich) treated Delta-TPG plate (Bioptics, Butler, PA) containing a 10ul drop of KSOM-BSA containing Hoechst 33342 (1ug/ml) and covered with pre-equilibrated mineral oil. The plate was mounted in a Chamlide (Quorum Technology, Inc. Guelph, ON, Canada) TC-L stage top environmental chamber to maintain temperature at 37°C and a CO₂ level of 5%. Imaging was performed with a 640 nm laser to detect the actin fenestrae labelled with SiR-actin.

Teneral (<24 hours old post emergence) male tsetse were fed on horse blood (TCS Biosciences, Buckingham, United Kingdom) and three days later were offered a second meal containing horse serum (to increase tissue visibility by removing red blood cell interference) with or without 500 ng/mL NTBC. Fifteen hours post-feeding, tsetse were anaesthetized on ice, and the midgut tissue (with attached proventriculi) was dissected into ice-cold PBS and immediately fixed in fresh 4% paraformaldehyde for 1 hour at room temperature (RT). Tsetse group survival rates were determined 72 hours post-NTBC administration. Fixed tissues were washed in PBS (and stained with SiR-actin (1,100 dilution, Cytoskeleton Inc.) for 3 hours at RT and posteriorly incubated in 500 ng/mL DAPI for 10 minutes at RT. Tissues were finally suspended in 1% (w/v) low-melting agarose at approximately 40°C mixed with Slowfade Diamond oil (Molecular Probes, Eugene, United States) on a slide. Slides were imaged using a Zeiss LSM-880 confocal laser scanning microscope and tissues were 3D-reconstructed from a series of z-stacks at intervals of 1.3 μm (Zeiss Company, Oberkochen, Germany).

All chemicals were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO) except otherwise indicated. SiR-actin was obtained from Cytoskeleton (Denver, CO) and FM4-64 was purchased from Thermo Fisher Scientific (Waltham, MA).