A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

The novel antibiotic rhodomyrtone traps membrane proteins in vesicles with increased fluidity.

The acylphloroglucinol rhodomyrtone is a promising new antibiotic isolated from the rose myrtle Rhodomyrtus tomentosa, a plant used in Asian traditional medicine. While many studies have demonstrated its antibacterial potential in a variety of clinical applications, very little is known about the mechanism of action of rhodomyrtone. Preceding studies have been focused on intracellular targets, but no specific intracellular protein could be confirmed as main target. Using live cell, high-resolution, and electron microscopy we demonstrate that rhodomyrtone causes large membrane invaginations with a dramatic increase in fluidity, which attract a broad range of membrane proteins. Invaginations then form intracellular vesicles, thereby trapping these proteins. Aberrant protein localization impairs several cellular functions, including the respiratory chain and the ATP synthase complex. Being uncharged and devoid of a particular amphipathic structure, rhodomyrtone did not seem to be a typical membrane-inserting molecule. In fact, molecular dynamics simulations showed that instead of inserting into the bilayer, rhodomyrtone transiently binds to phospholipid head groups and causes distortion of lipid packing, providing explanations for membrane fluidization and induction of membrane curvature. Both its transient binding mode and its ability to form protein-trapping membrane vesicles are unique, making it an attractive new antibiotic candidate with a novel mechanism of action.

VPS35 depletion does not impair presynaptic structure and function.

The endosomal system is proposed as a mediator of synaptic vesicle recycling, but the molecular recycling mechanism remains largely unknown. Retromer is a key protein complex which mediates endosomal recycling in eukaryotic cells, including neurons. Retromer is important for brain function and mutations in retromer genes are linked to neurodegenerative diseases. In this study, we aimed to determine the role of retromer in presynaptic structure and function. We assessed the role of retromer by knocking down VPS35, the core subunit of retromer, in primary hippocampal mouse neurons. VPS35 depletion led to retromer dysfunction, measured as a decrease in GluA1 at the plasma membrane, and bypassed morphological defects previously described in chronic retromer depletion models. We found that retromer is localized at the mammalian presynaptic terminal. However, VPS35 depletion did not alter the presynaptic ultrastructure, synaptic vesicle release or retrieval. Hence, we conclude that retromer is present in the presynaptic terminal but it is not essential for the synaptic vesicle cycle. Nonetheless, the presynaptic localization of VPS35 suggests that retromer-dependent endosome sorting could take place for other presynaptic cargo.

Megalencephalic leukoencephalopathy with cysts: the Glialcam-null mouse model.

OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic infantile-onset disease characterized by macrocephaly and white matter edema due to loss of MLC1 function. Recessive mutations in either MLC1 or GLIALCAM cause the disease. MLC1 is involved in astrocytic volume regulation; GlialCAM ensures the correct membrane localization of MLC1. Their exact role in brain ion-water homeostasis is only partly defined. We characterized Glialcam-null mice for further studies. METHODS: We investigated the consequences of loss of GlialCAM in Glialcam-null mice and compared GlialCAM developmental expression in mice and men. RESULTS: Glialcam-null mice had early-onset megalencephaly and increased brain water content. From 3 weeks, astrocytes were abnormal with swollen processes abutting blood vessels. Concomitantly, progressive white matter vacuolization developed due to intramyelinic edema. Glialcam-null astrocytes showed abolished expression of MLC1, reduced expression of the chloride channel ClC-2 and increased expression and redistribution of the water channel aquaporin4. Expression of other MLC1-interacting proteins and the volume regulated anion channel LRRC8A was unchanged. In mice, GlialCAM expression increased until 3 weeks and then stabilized. In humans, GlialCAM expression was highest in the first 3 years to then decrease and stabilize from approximately 5 years. INTERPRETATION: Glialcam-null mice replicate the early stages of the human disease with early-onset intramyelinic edema. The earliest change is astrocytic swelling, further substantiating that a defect in astrocytic volume regulation is the primary cellular defect in MLC. GlialCAM expression affects expression of MLC1, ClC-2 and aquaporin4, indicating that abnormal interplay between these proteins is a disease mechanism in megalencephalic leukoencephalopathy with cysts.

Mice with megalencephalic leukoencephalopathy with cysts: a developmental angle.

OBJECTIVE: Megalencephalic leukoencephalopathy with cysts (MLC) is a genetic disease characterized by infantile onset white matter edema and delayed onset neurological deterioration. Loss of MLC1 function causes MLC. MLC1 is involved in ion-water homeostasis, but its exact role is unknown. We generated Mlc1-null mice for further studies. METHODS: We investigated which brain cell types express MLC1, compared developmental expression in mice and men, and studied the consequences of loss of MLC1 in Mlc1-null mice. RESULTS: Like humans, mice expressed MLC1 only in astrocytes, especially those facing fluid-brain barriers. In mice, MLC1 expression increased until 3 weeks and then stabilized. In humans, MLC1 expression was highest in the first year, decreased, and stabilized from approximately 5 years. Mlc1-null mice had early onset megalencephaly and increased brain water content. From 3 weeks, abnormal astrocytes were present with swollen processes abutting fluid-brain barriers. From 3 months, widespread white matter vacuolization with intramyelinic edema developed. Mlc1-null astrocytes showed slowed regulatory volume decrease and reduced volume-regulated anion currents, which increased upon MLC1 re-expression. Mlc1-null astrocytes showed reduced expression of adhesion molecule GlialCAM and chloride channel ClC-2, but no substantial changes in other known MLC1-interacting proteins. INTERPRETATION: Mlc1-null mice replicate early stages of the human disease with early onset intramyelinic edema. The cellular functional defects, described for human MLC, were confirmed. The earliest change was astrocytic swelling, substantiating that in MLC the primary defect is in volume regulation by astrocytes. MLC1 expression affects expression of GlialCAM and ClC-2. Abnormal interplay between these proteins is part of the pathomechanisms of MLC.