Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT)

Abstract

Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton.microtubules | septins | actin | bacterial toxin | Clostridium difficile

Septins and Bacterial Infection

“…When exposing cells to CDT, the cellular cortex undergoes extensive remodeling, resulting in patches of actin depolymerization. Complexes of SEPT2-SEPT6-SEPT7-SEPT9 are recruited to the plasma membrane shortly after initial depolymerization events and form collar-like structures that initiate CDT-dependent protrusions (Nölke et al, 2016) (Figure 2B). Depletion of septins by siRNA or their inhibition with forchlorfenuron reduced protrusion formation, while septin overexpression increased protrusion emergence.…”

“…Depletion of septins by siRNA or their inhibition with forchlorfenuron reduced protrusion formation, while septin overexpression increased protrusion emergence. At the protrusion base, septins interact with the GTPase Cell Division Control Protein 42 (CDC42) and its effector Binder of Rho GTPase (BORG) (Nölke et al, 2016). These results are consistent with previous work using non-infected HeLa cells showing the regulation of septin assembly by BORGs (Joberty et al, 2001).…”

“…Septins have high affinity to End Binding 1 (EB1), a protein which associates to the plus end of microtubules, suggesting that septins guide microtubule polymerization in the extending protrusion (Nölke et al, 2016). The discovery that septins mediate microtubule apical guidance can have broad implications in cell biology.…”

The Unsolved Problem of How Cells Sense Micron-Scale Curvature

“…Septin filaments can be arranged to a variety of higher-order structures, some of which are at sites of micron-scale membrane curvature, including at the bud-neck in S. cerevisiae , and at the bases of dendritic spines and hyphal branches in neurons and filamentous fungi, respectively [22, 34–36]. At these sites, septins coordinate cell cycle progression [22], influence the organization of actin [37] and microtubules [38], and may function as a lateral diffusion barrier in the membrane [39–41]. The fact that septin structures are often localized to sites of micron-scale membrane curvature suggested that the septins might be able to “sense” these shallow curvatures.…”

Uncovering the Roles of Septins in Cilia

“…As well, SEPT7 was shown to interact with Rab8 (Dash et al, 2014), a small GTPase that functions in vesicle trafficking and ciliogenesis (Nachury et al, 2007; Yoshimura et al, 2007). Septins could also affect vesicle traffic via the cytoskeleton since they associate with microtubules and guide the direction of microtubule growth (Bowen et al, 2011; Nölke et al, 2016) and may control vesicular transport along them (Spiliotis et al, 2008). …”