Figure 4. DivIVA and an intact cell wall promote membrane domain maintenance.

(A) Lysates from MurG-Dendra2-expressing M. smegmatis spheroplasts (Melzer et al., 2018) or from the DivIVA-eGFP-ID strain depleted (off) or not (on) of DivIVA (Meniche et al., 2014) were sedimented in a sucrose density gradient. (B) DivIVA was depleted or not from mCherry-GlfT2-expressing M. smegmatis. Depletion of DivIVA delocalizes mCherry-GlfT2. M. smegmatis expressing MurG-Dendra2 (C) or coexpressing mCherry-GlfT2 and DivIVA-eGFP-ID (D) were spheroplasted or not (control) and imaged. In spheroplasted cells, the IMD-associated proteins distribute along the cell periphery. Arrows mark spheroplasts outside and within insets, which have increased size and brightness. Merged images correspond to fluorescent image with the corresponding phase contrast. (E) Model for self-organization of plasma membrane and cell wall in M. smegmatis. Brown line indicates the cell wall. Short brown lines perpendicular to the membrane and cell wall indicate that the cell wall is likely to be physically connected to the membrane in the PM-CW regions (Morita et al., 2005). All scale bars, 5 µm.

Figure 4—figure supplement 1. An intact cell wall supports MurG partitioning within the membrane.

M. smegmatis expressing MurG-Dendra2 was spheroplasted as in Figure 4A and then lysed by nitrogen cavitation. Lysates were separated by sucrose density gradient MurG-Dendra2 was visualized by in-gel fluorescence. MurG-Dendra2 distributes widely across the membrane fractions (compare to Figure 1C and quantitation in Figure 1—figure supplement 4A), consistent with the expanded distribution of cellular material in Figure 4A.