Lei Lu

We previously showed that transmembrane proteins laterally partition within Golgi cisternae: trafficking components (e.g., GPP130, a type II transmembrane protein) are enriched at the cisternal rim, whereas Golgi enzymes localize to the cisternal interior. However, the underlying mechanism was unclear. We found that the luminal juxtamembrane coiled coil of GPP130 is necessary and sufficient for rim localization, with coiled coil length, rather than sequence identity, determining partitioning. When retargeted to the ER, GPP130 preferentially localized to ER sheet rims and was partially excluded from the nuclear envelope, an exclusion reduced upon nuclear envelope expansion. Cryo-electron tomography revealed that the intraluminal widths of Golgi cisternal interiors, some ER sheets, and the nuclear envelope are smaller than the predicted coiled coil length. Two additional type II Golgi transmembrane proteins with long luminal juxtamembrane coiled coils similarly localized to cisternal rims. These findings identify intraluminal width as a mechanism governing the lateral partitioning of type II transmembrane proteins with long luminal coiled coils.

The Golgi apparatus is a central hub for protein trafficking and signaling, yet its rapid imaging and cell-selective disruption remain challenging. Here, we report cycling molecular assemblies (CyMA) for fast Golgi imaging and cell-selective interference. CyMA precursors are acetylated amphiphilic thiopeptides that traverse plasma membrane and are deacetylated by intracellular thioesterases. This exposes thiols that undergo palmitoylation by Golgi-resident palmitoyl acyltransferases utilizing palmitoyl-CoA. The resulting palmitoylated peptides self-assemble into dynamic nanostructures (i.e., CyMA) localized at the Golgi. Their continuous, reversible S -acylation enables near-instantaneous Golgi imaging. Replacing fluorophore with a biphenyl motif promotes CyMA accumulation and disrupts functions such as protein modifications, trafficking, and secretion, leading to cell death. This study establishes dynamic supramolecular assembly as an active and selective strategy for Golgi-targeting, pleiotropically interfering with Golgi functions, which may be applicable to targeting other organelles by utilizing alternative enzyme switches to enable kinetic trapping.

The Golgi is a eukaryotic organelle that modifies, sorts, and transports proteins and lipids to their destinations. In mammals, the Golgi is a complex network of interconnected stacks, with each stack made up of closely packed flat membrane sacs called cisternae. The Golgi remains a poorly understood organelle, fraught with controversies despite decades of research. The challenge of visually resolving the cisternal organization of the Golgi under light or electron microscopy contributes to its enigmatic nature. We have developed new imaging methods, including GLIM, en face averaging, and side averaging, to resolve the nocodazole-induced Golgi ministacks. These tools provide new insights into the organization and functions of the Golgi. They enabled us to rapidly and quantitatively investigate cisternal Golgi organization and intra-Golgi transport of secretory cargos. New insights have been revealed about the spatial distribution of Golgi proteins involved in processing and trafficking, the differential partitioning of secretory cargos within cisternae based on their sizes, and the specific Golgi exit sites for constitutive versus signal-dependent secretory cargos. Additionally, the intra-Golgi transport kinetic data acquired from these tools provide a basis for evaluating different intra-Golgi transport models.

Drug resistance in bacteria is a major problem that calls for new classes of antimicrobial drugs. We report a biodegradable poly(imidazolium ester) (PIE), P8, with excellent broad-spectrum antibacterial activity, high therapeutic selectivity, and an unexploited mechanism of action. P8, a short oligomer, translocates across bacterial membrane and phase separates with intracellular nucleic acids, forming biomolecular condensates. P8 binds the DNA minor groove and intercalates with DNA, interacting with it via electrostatic and hydrogen-bonding interactions. The phase separation of nucleic acids modulated by P8 inhibits in vitro transcription, thereby impeding translation and potentially leading to cell death. Bacterial cytological profiling indicates that the antimicrobial mechanism of P8 differs from those of conventional antibiotics, though it also suggests that P8 may inhibit RNA synthesis. P8 is safe and effective against drug-resistant bacteria in murine models of systemic, intramuscular, and lung infections. This study shows the great potential of intracellular biomolecular condensate formation for combating drug-resistant bacteria.

Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to SMO-mediated hedgehog stimulation. Our work demonstrates the critical requirement of ARL13B for ciliogenesis and hedgehog signaling, at least in cultured cells, and suggests that ARL13B plays a more crucial role in ciliary function than previously understood.

How the intra-Golgi secretory transport works remains a mystery. The cisternal progression and the stable compartment models have been proposed and are under debate. Classic cisternal progression model posits that both the intra-Golgi transport and Golgi exit of secretory cargos should occur at a constant velocity dictated by the cisternal progression; furthermore, COPI-mediated intra-Golgi retrograde transport is essential for maintaining the Golgi organization. Leveraging our recently developed Golgi imaging tools in nocodazole-induced Golgi ministacks, we found that the intra-Golgi transport velocity of a secretory cargo decreases during their transition from the cis to the trans -side of the Golgi, and different cargos exhibit distinct velocities even within the same cisternae. We observed a vast variation in the Golgi residence times of different cargos. Remarkably, truncation of the luminal domain causes the Golgi residence time of Tac — a standard transmembrane secretory cargo without intra-Golgi recycling signals — to extend from 16 min to a notable 3.4 hr. Additionally, when COPI-mediated intra-Golgi retrograde transport was inhibited by brefeldin A, we found that nocodazole-induced Golgi can remain stacked for over 30–60 min. Therefore, our findings challenge the classical cisternal progression model and suggest the stable compartment nature of the Golgi.

How the intra-Golgi secretory transport works remains a mystery. The cisternal progression and the stable compartment models have been proposed and are under debate. Classic cisternal progression model posits that both the intra-Golgi transport and Golgi exit of secretory cargos should occur at a constant velocity dictated by the cisternal progression; furthermore, COPI-mediated intra-Golgi retrograde transport is essential for maintaining the Golgi organization. Leveraging our recently developed Golgi imaging tools in nocodazole-induced Golgi ministacks, we found that the intra-Golgi transport velocity of a secretory cargo decreases during their transition from the cis to the trans-side of the Golgi, and different cargos exhibit distinct velocities even within the same cisternae. We observed a vast variation in the Golgi residence times of different cargos. Remarkably, truncation of the luminal domain causes the Golgi residence time of Tac — a standard transmembrane secretory cargo without intra-Golgi recycling signals — to extend from 16 min to a notable 3.4 h. Additionally, when COPI-mediated intra-Golgi retrograde transport was inhibited by brefeldin A, we found that nocodazole-induced Golgi can remain stacked for over 30 - 60 minutes. Therefore, our findings challenge the classical cisternal progression model and suggest the stable compartment nature of the Golgi.

Small G protein ARL13B localizes to the cilium and plays essential roles in cilium biogenesis, organization, trafficking, and signaling. Here, we established multiple ARL13B knockout cell lines using the CRISPR/Cas9 system. Surprisingly, all our cell lines lost their cilia completely, in contrast to the reported short cilium and reduced ciliogenesis phenotype. We found that multiple regions of ARL13B are necessary for a complete rescue. Additionally, we found that ARL13B knockout cells also lost their response to SMO-mediated hedgehog stimulation. Our work demonstrates the critical requirement of ARL13B for ciliogenesis and hedgehog signaling, at least in cultured cells, and suggests that ARL13B plays a more crucial role in ciliary function than previously understood.

The Golgi apparatus is a critical organelle responsible for intracellular trafficking and signaling, orchestrating essential processes such as protein and lipid sorting ^1–5 . Dysregulation of its function has been implicated in various pathologies, including obesity, diabetes, and cancer, highlighting its importance as a potential therapeutic target. Despite this, the development of tools to selectively target the Golgi in specific cell types remain a significant unmet challenge in imaging and drug discovery. Golgi-specific enzyme activities, such as those mediated by protein acyltransferases and thioesterases ⁶ , offer an untapped opportunity to develop subcellularly localized therapeutics. Current approaches predominantly rely on direct protein binding but lack the necessary cell selectivity ⁷ , underscoring the unmet need for innovative strategies to selectively disrupt Golgi function in cancer cells. Here, we report the development of cycling molecular assemblies (CyMA), a novel class of small peptide derivatives (e.g., dipeptides), which exploit the unique enzymatic environment of the Golgi to establish futile cycles of reversible S-acylation. These assemblies selectively accumulate in cancer cell Golgi, interfering with protein S-acylation cycles and disrupting organelle homeostasis. CyMA impair key Golgi functions, including protein trafficking, glycosylation, and secretion, while demonstrating selective sparing hepatocytes and immune cells such as M1 macrophages. This selective activity represents a paradigm shift, utilizing an enzyme switch and leveraging intracellular environment rather than direct protein binding. Unlike conventional approaches, CyMA reduce tumor growth, drug resistance, and metastasis by pleiotropically disrupting Golgi related functions. By demonstrating the potential of futile cycles as a therapeutic strategy ⁸ , this study introduces a generalizable method for targeting organelle-specific enzyme activities. These findings not only underscore the therapeutic potential of CyMA in cancer but also pave the way for future applications in other Golgi-associated diseases.