Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin

Karasmanis, Eva; Phan, Cat Thi; Angelis, Dimitrios; Kesisova, Ilona A.; Hoogenraad, Casper C.; McKenney, Richard J.; Spiliotis, Elias T.

doi:10.1016/j.devcel.2018.08.004

Cited by 29 publications

(55 citation statements)

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“…We tested if SEPT9 impacts lysosome traffic in primary rat embryonic hippocampal neurons. In early stages of neuronal morphogenesis, SEPT9 is present in the developing axon before it becomes enriched on dendritic microtubules (Karasmanis et al, 2018). In the axons of these cells, which consist of unidirectional plus endout oriented microtubules, we found that SEPT9 was enriched more than two-fold in LAMP1positive endolysosomes compared to Rab5-containing early endosomes (Fig.…”

Section: Introductionmentioning

confidence: 68%

“…This scaffolding function may facilitate cooperative assembly of dynein-dynactin into the nanodomains of motor teams, which have been observed on lipid microdomains and microtubules (Cella Zanacchi et al, 2019;Rai et al, 2016) Among the members of the septin family of GTPases, SEPT9 is unique in possessing a faster GTPase activity and a dimerization interface, which is characterized by nucleotide-dependent plasticity (Castro et al, 2020;Zent and Wittinghofer, 2014). Additionally, SEPT9 has been reported to function and localize independently of its heteromeric partners as the central subunit of the octameric SEPT2/6/7/9 complex (Estey et al, 2010;Karasmanis et al, 2018). Our results indicate that SEPT9 interacts with dynein independently of SEPT2/6/7, but do not rule out the possibility that SEPT9 functions in a complex with SEPT2/6/7 on lysosomal membranes.…”

Section: Discussionmentioning

confidence: 99%

“…Plasmids encoding for GFP-EEA1 was a gift from Silvia Corvera ( The plasmids GFP-Rab5A (Dolat and Spiliotis, 2016), mCherry-N1-humanSEPT9_i1 (Bai et al, 2013), mCherry-N1-SEPT2 (Bowen et al, 2011), pmCherry-N1-ratSEPT9 and pET28a-mCherry-humanSEPT9 (Karasmanis et al, 2018), pET28a-SEPT9(i1), pET28a-SEPT9-NTE (1-283 aa) and pET28a-SEPT9G (283-586 aa) (Bai et al, 2016) were constructed as previously described. The pnEA-vH and pnCS vectors encoding for His-SEPT2 and SEPT6/7-strep, respectively, have been described previously .…”

Section: Plasmids and Constructsmentioning

confidence: 99%

“…Primary antibodies were incubated overnight at 4 o C and secondary antibodies were incubated at room temperature for 1 h in 0.2% porcine gelatin BSA and 0.1% Saponin in PBS. Primary rat hippocampal neurons were fixed and stained as previously described (Karasmanis et al, 2018). Fig.…”

Section: Immunoprecipitations and Binding Assaysmentioning

confidence: 99%

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A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport

Kesisova

Robinson

Spiliotis

2020

Preprint

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The intracellular position of lysosomes is critical for cell metabolism and signaling. Kesisova et al discovered a membrane-associated septin GTPase scaffold of dynein-dynactin that promotes retrograde traffic and perinuclear lysosome clustering at steady state and in response to oxidative stress. Condensed title: A septin GTPase mechanism of dynein-driven traffic Abbreviations: amino acids, aa; dynein heavy chain, DHC; dynein intermediate chain, DIC, dynein light intermediate chain, DLIC; early endosome antigen 1, EEA1; early sorting complex required for transport, ESCRT; G-domain, GTPase domain; guanosine-5′-triphosphate, GTP; guanosine diphosphate, GDP; guanosine 5′-O-[γ-thio]triphosphate, GTPγS; JNK-interaction protein-1, JIP-1; lysosomal associated membrane protein 1, LAMP1; late endosomal/lysosomal adaptor, MAPK and mTOR activator 4, LAMTOR4; multivesicular body, MVB; N-terminal extension, NTE; reactive oxygen species, ROS; septin, SEPT; tumor susceptibility gene 101, TSG101. AbstractThe metabolic and signaling functions of lysosomes depend on their intracellular positioning and trafficking, but the underlying mechanisms are little understood. Here, we have discovered a novel septin GTPase-based mechanism for retrograde lysosome transport. We found that septin 9 (SEPT9) associates with lysosomes, promoting the perinuclear localization of lysosomes in a Rab7-independent manner. SEPT9 targeting to mitochondria and peroxisomes is sufficient to recruit dynein and cause perinuclear clustering. We show that SEPT9 interacts with both dynein and dynactin through its GTPase domain and N-terminal extension, respectively. Strikingly, SEPT9 associates preferentially with the dynein intermediate chain (DIC) in its GDP-bound state, which favors dimerization and assembly into septin multimers. In response to oxidative cell stress induced by arsenite, SEPT9 localization to lysosomes is enhanced, promoting the perinuclear clustering of lysosomes. We posit that septins function as GDP-activated scaffolds for the cooperative assembly of dynein-dynactin, providing an alternative mechanism of retrograde lysosome transport at steady state and during cellular adaptation to stress. barriers, which control protein localization in a spatiotemporal specific manner (Bridges and Gladfelter, 2015;Caudron and Barral, 2009). In the endocytic pathway, septins associate preferentially with endolysosomes that are enriched with phosphatidylinositol 3,5-bisphosphate and Rab7 (Dolat and Spiliotis, 2016). Moreover, septins are critical for lysosome merging with macropinosomes and Shigella bacteria undergoing autophagy (Dolat and Spiliotis, 2016;Krokowski et al., 2018). In a proteomic study of the dynein interactome, septins were identified as potential binding partners of DIC, DLIC and the dynein adaptor BiCD2 (Redwine et al., 2017).Here, we report that membrane-associated septins provide a novel GDP-activated mechanism for the retrograde dynein-driven transport of lysosomes.

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Section: Introductionmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Plasmids and Constructsmentioning

confidence: 99%

Section: Immunoprecipitations and Binding Assaysmentioning

confidence: 99%

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A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport

Kesisova

Robinson

Spiliotis

2020

Preprint

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“…SEPT9 isoforms associate with the SEPT7 subunits of the hetero-hexameric SEPT7-SEPT6-SEPT2-SEPT2-SEPT6-SEPT7 complex, but longer SEPT9 isoforms might be preferred over shorter ones (14,15,20). Interestingly, in some cell types and processes, SEPT9 proteins appear to localize and function independently of SEPT2/6/7 (19,27,28).…”

Section: Introductionmentioning

confidence: 99%

Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells

Devlin

Perkins

Bowen

et al. 2019

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Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary significantly in their N-terminal extensions. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (e.g., nuclei, centrosomes, cilia) and functions such as cell signaling and ubiquitination. Notably, the interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear, signaling and ubiquitinating proteins. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer. L.D. performed experiments, analyzed data, made figures and co-wrote manuscript with E.T.S.; G.P. contributed technically and intellectually to ESI/LC-MS/MS experimentation and softwarebased peptide identification; J.R.B. performed microscopy imaging; C.M. created MCF-7 cell lines and contributed to the manuscript; E.T.S. conceived and directed the project (experiments and analyses), drafted manuscript and edited figures.

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Production and analysis of a mammalian septin hetero‐octamer complex

et al. 2020

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The septins are filament-forming proteins found in diverse eukaryotes from fungi to vertebrates, with roles in cytokinesis, shaping of membranes and modifying cytoskeletal organization. These GTPases assemble into rod-shaped soluble hetero-hexamers and hetero-octamers in mammals, which polymerize into filaments and higher order structures. While the cell biology and pathobiology of septins are advancing rapidly, mechanistic study of the mammalian septins is limited by a lack of recombinant hetero-octamer materials. We describe here the production and characterization of a recombinant mammalian septin hetero-octamer of defined stoichiometry, the SEPT2/SEPT6/SEPT7/ SEPT3 complex. Using a fluorescent protein fusion to the complex, we observed filaments assembled from this complex. In addition, we used this novel tool to resolve recent questions regarding the organization of the soluble septin complex. Biochemical characterization of a SEPT3 truncation that disrupts SEPT3-SEPT3 interactions is consistent with SEPT3 occupying a central position in the complex while the SEPT2 subunits are at the ends of the rod-shaped octameric complexes. Consistent with SEPT2 being on the complex ends, we find that our purified SEPT2/SEPT6/SEPT7/SEPT3 heterooctamer copolymerizes into mixed filaments with separately purified SEPT2/SEPT6/ SEPT7 hetero-hexamer. We expect this new recombinant production approach to lay essential groundwork for future studies into mammalian septin mechanism and function.

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Polarity of Neuronal Membrane Traffic Requires Sorting of Kinesin Motor Cargo during Entry into Dendrites by a Microtubule-Associated Septin

Cited by 29 publications

References 0 publications

A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport

A septin GTPase scaffold of dynein-dynactin motors triggers retrograde lysosome transport

Proteomic profiling of the oncogenic septin 9 reveals isoform-specific interactions in breast cancer cells

Production and analysis of a mammalian septin hetero‐octamer complex

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