The efficient delivery of lysosomes is essential for many cell functions, such as the degradation of unwanted intracellular components by autophagy and the killing and digestion of extracellular microbes within phagosomes. Using the amoeba Dictyostelium discoideum we find that cells lacking Katnip (Katanin interacting protein) have a general defect in lysosomal delivery and although they make autophagosomes and phagosomes correctly, cells are then unable to digest them.

Katnip is largely unstudied yet highly conserved across evolution. Previously studies found Katnip mutations in animals cause defects in cilia structure. Here we show that Katnip plays a more general role in maintaining microtubule function. We find that loss of Katnip has no overall effect on microtubule dynamics or organisation, but is important for the transport and degradation of endocytic cargos. Strikingly, Katnip mutants become highly sensitive to GFP-tubulin expression, which leads to microtubule tangles, defective anaphase extension and slow cell growth. Our findings establish a general role for Katnip in regulating microtubule function, beyond the previous roles described in cilia. We speculate this is via a key function in microtubule repair, required to maintain endosomal trafficking and lysosomal degradation.

Movie S1: Autophagosome formation in wild-type and katA−1 cells. Cells expressing GFP-Atg8aimaged by spinning disc after compression under agarose.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s04.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab1a013faa9142f073c56719441adc05b1fbf2d5f0a8a1157a8f9c0fffcae157592935816eba02fe2bd4dbbc4b798f2592a497fc15b18475f3b3695cfe936b2eb10eeb3e894085ed3c790dda04c0ddbd7310471

Movie S2: Photo-induced relocation of GFP-dlKatnip. Confocal movie of wild-type cells under continuous illumination on a spinning disc microscope.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s05.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c3b4a0b6b229f489ee0dcc53baba4c67f2d04950500208d0c8920a581fd30937fe948fed8cbded21d469cdb8fbaa6a7fa5cc998b9e08e6b05fc97a27e72d36be09bff874eb0236b42c784b87f4f1c85e194fb5213cb74e8

Movie S3: Photo-induced relocation of GFP-dlKatnip after 30 μM nocodazole treatment. Cells were images as in movie 2. Note the more dense localisation to the shortened microtubules.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s06.,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518511abb28bed2c6f7421a565d9f7a5b3e3f5a40bc644191f5cacfb3cc9bc55304dc1c46558ab6522739decd45c6dec6a8654b8369cdf994bf5e08d6d62d0c63d92507c3b4a0b6b229f489ee0dcc53baba4c67f2d04950500208d0c8920a581fd30937fe948fed8cbded21d469cd88fbaa6a7fa5cc9643b0940177e2318a6ff24985b9eadd9d90d0f0a9cb49779be929239734d300c4db9373428

Movie S4: Dynamics of GFP-α-tubulin in wild-type and katA−1 cells. Maximum intensity projection from a confocal timelapse.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s07.,1920,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518b17abb28b8c6166875a97f369b6d543b680497f5bca01d1632f390ebb6e08bd2a2725a70a05738b03ddc7251bffb1206bcd384e738aacf0abe9b4502e102dfa92763a3eb1af3ab0841f330457cf9cda7a0f8d35d8ee847f9b8c7aae2e66db3e9e4cff91826c30068c2af8c67aa42ac1acccd719e774eb9331ea69d4828e494ebb5ed2c8bb9526590828b576e4c46d2fe280c928343b166f38db5a1ea624009576886b1df1303a107e2bb8c2

Movie S5: Mitosis of wild-type cells, expressing GFP-α-tubulin.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s08.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab1a013faa9142f073c56719441adc05b1fbf2d5f0a8a1157a8f9c0fffcae157592935816eba02fe2bd4db7c4b798f2592a492514c0829d5781fe96356e06550f81e0efacd7a1e081d5afcfbdea19f75d751ab8

Movie S6: Mitosis of Katnip null cells expressing GFP-α-tubulin.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s09.,652,642,.mp4.m3u8?b92b4ad1b4f274c70877518515abb28bda92fbabe7b929571bd415190bf44d1790b7324fb90f184f91fab10e395327a1bef9725d0c99a358bbbe69c082a95f9d49957eb2932b2be9a6f328f5ec3ab1a013faa9142f073c56719441adc05b1fbf2d5f0a8a1157a8f9c0fffcae157592935816eba02fe2bd4db6c4b798f2592a49dbe7ceb3be1265564b02178fbc07719ecf71240dba06db1458f6828945967c0892

Movie S7: Example of phagosome movement within control and KatA−1 cells. Cells were allowed to engulf 1μm beads for 10 minutes prior to compression under agarose and imaging by DIC microscopy.

https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e22-02-0063/20221226/media/mc-e22-02-0063-s10.,1200,960,900,768,652,642,.mp4.m3u8?b92b4ad1b4f274c7087751841cabb28b19a3e23570e4794628d00a388fabd4f47061f4b4b81c11dac4c336cd5be6a2a6a495e5def8479aeedf32e6f4ec8f325c8354b92f9a15e63bba6a844857b798520906cd89a1b0f4b2b4cb69bbe7c5a3b8bbcc924a4b68886ed3278ba5aeaf3661bd5cd4983f6f29ea066e83ef4020f23ccb018f9f9c1633362880dd758a209a9b98fe9d370e120247379e2f585caa5ffacf600aade64e3b05af329a5aa01254700b96