Abstract:The septin family of proteins has fascinated cell biologists for decades due to the elaborate architecture they adopt in different eukaryotic cells. Whether they exist as rings, collars, or gauzes in different cell types and at different times in the cell cycle illustrates a complex series of regulation in structure. While the organization of different septin structures at the cortex of different cell types during the cell cycle has been described to various degrees, the exact structure and regulation at the f… Show more
“…The septin cytoskeleton, which is the fourth cytoskeletal element of eukaryotic cells, has a number of homeostatic and specialized functions in different tissues [1,[24][25][26]. In contrast to the extensive toolbox for probing the structure and dynamics of either actin cytoskeleton or microtubules, experimental tools to probe septin functions are limited.…”
Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.
“…The septin cytoskeleton, which is the fourth cytoskeletal element of eukaryotic cells, has a number of homeostatic and specialized functions in different tissues [1,[24][25][26]. In contrast to the extensive toolbox for probing the structure and dynamics of either actin cytoskeleton or microtubules, experimental tools to probe septin functions are limited.…”
Septins are GTP-binding proteins that self-assemble into high-order cytoskeletal structures, filaments, and rings. The septin cytoskeleton has a number of cellular functions, including regulation of cytokinesis, cell migration, vesicle trafficking, and receptor signaling. A plant cytokinin, forchlorfenuron (FCF), interacts with septin subunits, resulting in the altered organization of the septin cytoskeleton. Although FCF has been extensively used to examine the roles of septins in various cellular processes, its specificity, and possible off-target effects in vertebrate systems, has not been investigated. In the present study, we demonstrate that FCF inhibits spontaneous, as well as hepatocyte growth factor-induced, migration of HT-29 and DU145 human epithelial cells. Additionally, FCF increases paracellular permeability of HT-29 cell monolayers. These inhibitory effects of FCF persist in epithelial cells where the septin cytoskeleton has been disassembled by either CRISPR/Cas9-mediated knockout or siRNA-mediated knockdown of septin 7, insinuating off-target effects of FCF. Biochemical analysis reveals that FCF-dependent inhibition of the motility of control and septin-depleted cells is accompanied by decreased expression of the c-Jun transcription factor and inhibited ERK activity. The described off-target effects of FCF strongly suggests that caution is warranted while using this compound to examine the biological functions of septins in cellular systems and model organisms.
“…However, how these are organized into the CR as a whole is unclear. Other than septins, which form filaments on the plasma membrane and bind components of the CR (Marquardt et al, 2018), most other CR components have not been detected by EM. Super-resolution microscopy can probe their organization at the nanoscale level and two such studies have been performed in S. pombe (Laplante et al, 2016;McDonald et al, 2017).…”
Animal cells, amoebas and yeast divide using a force-generating, actin-and myosin-based contractile ring or 'cytokinetic ring' (CR). Despite intensive research, questions remain about the spatial organization of CR components, the mechanism by which the CR generates force, and how other cellular processes are coordinated with the CR for successful membrane ingression and ultimate cell separation. This Review highlights new findings about the spatial relationship of the CR to the plasma membrane and the arrangement of molecules within the CR from studies using advanced microscopy techniques, as well as mechanistic information obtained from in vitro approaches. We also consider advances in understanding coordinated cellular processes that impact the architecture and function of the CR.
“…A critical step in the PBS assembly involves the recruitment of the septins. The four septin-units Cdc11, Cdc12, Cdc3 and Cdc10 form octameric rods that polymerize at the bud neck into filaments and higher-order structures like rings and collars (Bertin et al, 2008;Marquardt et al, 2019). Cdc11 is the subunit that caps the rod at its both ends and thus plays a critical role in filament formation (Garcia et al, 2011;Brausemann et al, 2016).…”
Section: Introductionmentioning
confidence: 99%
“…Cdc11 is the subunit that caps the rod at its both ends and thus plays a critical role in filament formation (Garcia et al, 2011;Brausemann et al, 2016). During mitosis the septins split into two rings and enclose the space where cytokinesis and abscission occurs (Marquardt et al, 2019). After abscission septins are disassembled from the site of cytokinesis and transferred in a Cdc42 GTP -dependent manner to the new PBS (Gladfelter et al, 2002;Caviston et al, 2003).…”
Section: Introductionmentioning
confidence: 99%
“…The ring remains at the base of the bud during further membrane and cell wall growth and restricts the free movement of the plasma membrane between mother and daughter cell (Barral et al, 2000). The mechanisms of septin recruitment and assembly are not fully understood (Marquardt et al, 2019). A central role is played by the PAK Cla4 and the paralogous proteins Gic1 and Gic2 (Kadota et al, 2004).…”
Yeast cells select at the beginning of each cell cycle the position of their new bud. The recruitment of the septins to this prospective bud site (PBS) is one of the critical events in a complex assembly pathway that culminates in the outgrowth of a new daughter cell. The septin-rods follow hereby the high concentration of Cdc42 GTP that is generated by the focused location of its GEF Cdc24. We show that Cdc24 not only activates Cdc42 but temporarily interacts shortly before budding with Cdc11, the subunit that caps septin rods at its both ends. Mutations in Cdc24 that reduce the affinity to Cdc11 impair septin assembly and decrease the stability of the polarity patch. The interaction between septins and Cdc24 thus reinforces bud assembly at sites where septin structures are formed. Once the septins polymerize into the ring, Cdc24 transfers to its center and directs from there the further outgrowth of the membrane.
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