Skip to main content
. Author manuscript; available in PMC: 2024 Oct 9.
Published in final edited form as: Dev Cell. 2023 Aug 7;58(19):1917–1932.e6. doi: 10.1016/j.devcel.2023.07.009

Figure 3: Midbody proteins and RNAs behave as ribonucleoprotein granules.

Figure 3:

(A) Synchronized HeLa cells (n=10) were treated at the MB stage for 90 seconds with 1,6-hexanediol and then were allowed to recover in normal medium for specified times (T = minutes post-hexanediol). The MB kinesin MKLP1 protein dispersed upon hexanediol addition, reforming spatially disseminated aggregates over time that surrounded the bridge in projected Z-series images. The MB structural component alpha-tubulin was unaffected by hexanediol treatment.

(B) Treatment with 1,6-hexanediol (hex) also affected polyA localization at the dark zone (n=7). We observed a loss of polyA and dissolution of the MKLP1 signal in the intercellular bridge.

(C) Live imaging of hexanediol-treated HeLa cells expressing a GFP-MKLP1 fusion protein and incubated with fluorescent SiR-tubulin (red) revealed a rapid and sustained partial loss (30% decrease) of MKLP1 levels at the native MB location; in contrast, the closely related mitotic kinesin MKLP2 fused to GFP exhibited no change in intensity after hexanediol treatment. The 30% loss of MKLP1-GFP after hexanediol treatments reveals that this kinesin is specifically sensitive to 1,6-hexanediol.

(D) FRAP analysis of GFP-MKLP1 MBs showed no recovery after photobleaching, suggesting little mobility of GFP-MKLP1 within the MB granule in native MBs.

(E-H) A functional range of MB matrix proteins (ANXA11, ARC, TDP-43, and TIA1) dispersed and reaggregated in apposition to MKLP1 upon hexanediol treatment (T=0 seconds) and after a long recovery time (T=30 seconds)(n=10 for E-H). Interestingly, all hexanediol-sensitive components tested reaggregated in domains complementary, but tightly apposed, to MKLP1. Of note, we often observed that only a portion of MB factors moves farther away from their original location in the intercellular bridge after hexanediol treatment. For example, the bulk of TIA1 remained diffuse in the dark zone immediately after treatment, but TIA1 quickly assembled back to its normal localization pattern after 30 minutes. MB expression in untreated controls was similar to MKLP1 for all hexanediol-sensitive MB factors (Fig. 4A, B). See also Fig. 4 for a timed series of hexanediol-mediated dissolution and reaggregation of RacGAP, TIA1, ANXA11, and ARC.

Scale bars are 1 μm.