Molecular mechanisms of membrane remodeling

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Sub-Project 1. Cytoskeletal control of secretion-associated membrane remodeling. In epithelial tissues specialized for secretion, proteins are compartmentalized within large secretory granules (SGs) that undergo fusion with the plasma membrane (PM) to discharge their contents. Following fusion, the SG membranes must incorporate into the PM, requiring extensive reshaping. We determined that this process involves the assembly of a force-producing scaffold composed of filamentous actin and two isoforms of non-muscle myosin II (NMIIA and NMIIB). These structures dynamically encase the SG membrane and coordinate its integration into the PM. Our objectives were to elucidate the mechanical forces generated by this actomyosin complex and identify the molecular components that regulate its recruitment. By employing ISMIC, we revealed that the membrane integration process unfolds in two mechanistically distinct phases. The first involves stabilization by an NMIIA-actin lattice network, with actin polymerization guided by the formin mDia1. Subsequently, a second module comprising branched actin filaments, assembled through Arp2/3 activation and linked to membranes via Ezrin, exerts integration forces. Perturbation of either module leads to marked defects: mDia1 disruption results in unregulated SG expansion, whereas Arp2/3 or Ezrin loss delays membrane incorporation. These findings delineate a sequential cytoskeletal program responsible for remodeling fused SG membranes. Live-cell dynamics further revealed Ezrin's role as a linker, tethering branched actin to the membrane interface under tension. Additionally, we observed that three septin family GTPases-SEPT2, SEPT6, and SEPT7-form, surprisingly, a distinct set of lattices around SGs. Genetic loss of SEPT7 impaired F-actin organization without disrupting NMIIA assembly, while inhibition of SEPT2 blocked integration through NMIIA suppression. Our data suggest that septins act as spatial organizers, enabling the coordinated activity of cytoskeletal elements during secretion. The modular cooperation of NMIIA, formins, Arp2/3, Ezrin, and septins represents a finely tuned force-generating architecture regulating granule integration. Future experiments will explore the recruitment dynamics of these components using real-time super-resolution microscopy in vivo. Sub-Project 2. Dynamic cytoskeletal remodeling during neutrophil migration. To evaluate whether the mechanisms of membrane remodeling identified during secretion are reused in other processes, we investigated migration in immune cells such as neutrophils-key effectors of inflammation. Using in vivo imaging, we characterized neutrophil behavior during transendothelial migration and interstitial crawling. In contrast to simplified models, we observed that NMIIA is prominently recruited to the leading edge during active migration, assembling into 3D lattices structurally analogous to those seen during SG integration but independent of F-actin. We found that the organization of these NMIIA structures depends on PI3K signaling pathways (rather than the canonical RhoA mechanism observed in 2D systems), although mini-filament assembly remains unaffected. Functionally, NMIIA contributes to protrusion stabilization and the maintenance of migratory directionality. It also facilitates efficient transmigration across epithelial barriers. To understand these dynamics quantitatively, we developed machine learning pipelines to analyze protrusion life cycles, curvature, and cytoskeletal distribution. Persistent protrusions exhibited higher NMIIA occupancy and unique geometric configurations. These data suggest that NMIIA encodes spatial memory and reinforces front-rear polarity during immune surveillance. These mechanisms may also underlie the migratory persistence of tumor-infiltrating immune cells or metastatic cancer cells. We have also identified molecular intermediates downstream of PI3K that control NMIIA lattice formation using immunoprecipitation and mass spectrometry. In parallel, we established protocols to reconstruct the ultrastructure of these lattices through correlative ISMic and focused ion beam scanning electron microscopy (FIB-SEM). Together, these results position NMIIA lattices as critical architectural elements for directional guidance in confined tissue environments. Sub-Project 3. Membrane trafficking, immune evasion, and metabolic reprogramming during tumor evolution. To investigate how membrane dynamics influence neoplastic transformation, we implemented long-term intravital imaging in a mouse model of head and neck squamous cell carcinoma (HNSCC) induced by the tobacco-mimetic carcinogen 4-nitroquinoline 1-oxide (4NQO). Using fluorescent reporter mice, we monitored the evolution of premalignant and malignant lesions in the tongue. Surprisingly, only 25% of dysplastic lesions progressed to carcinoma, with the remainder regressing spontaneously. This heterogeneity allowed us to identify key determinants of progression. We developed an AI-based framework for 3D segmentation, unbiased tumor detection, and correlation of lesion fate with cellular morphology, NADH-derived metabolic state, cytoskeletal architecture (F-actin, NMIIA), and extracellular matrix remodeling (collagen I). Progressive lesions exhibited spatial metabolic polarization, with suppressed glycolysis centrally and enhanced activity at the invasive edge. In contrast, regressing lesions displayed transient metabolic reductions and activation of autophagy. Genetic ablation of Atg7 confirmed a growth-suppressive role of autophagy in early tumor phases. Immune cell profiling revealed preferential recruitment of myeloid cells to nascent lesions. These cells infiltrated metabolically suppressed tumors and adopted distinct morphologies, suggesting a shift from surveillance to immunosuppressive behavior. Concurrently, we analyzed EGFR signaling, a key driver in HNSCC. Activation initiated in epithelial patches and intensified in mesenchymal-like cells at invasive margins. EGFR signaling was regulated by Rab25, a small GTPase previously shown to be downregulated in HNSCC. Moreover, we recently demonstrated that EGFR activation triggers rapid removal of terminal sialic acids from surface glycoproteins via the neuraminidases Neu1 and Neu3. This process, termed GlycoSwitch (GS), unmasks galectin-binding motifs, allowing galectin-3 to cluster glycoproteins for clathrin-independent endocytosis. Notably, this switch is acutely pH-sensitive and involves the Na+/H+ exchanger NHE1, which acidifies the pericellular environment and enables the activation of Neu1 and Neu3. This acidification-dependent triggering step ensures that glycan remodeling can occur rapidly and locally at the membrane in response to growth factor stimulation, such as EGFR signaling. The internalized cargo is remodeled in the Golgi and returned to polarized plasma membrane regions. We now propose that in tumors, GS shifts the glycosylation code to cluster immunosuppressive cues at the cell surface. This reprogramming may promote evasion via sialic acid-binding immunoglobulin-type lectins (Siglecs), which dampen immune activation. The GS pathway thus represents a fast-acting, EGFR-dependent mechanism linking oncogenic signaling to immune escape and spatial trafficking control in epithelial cancers. The integration of pH modulation, neuraminidase activity, and galectin engagement underscores the coordination between signaling cues and surface glycome dynamics. We will explore how this mechanism interfaces with nutrient gradients, hypoxia, and immune cell localization. In the next cycle, we aim to dissect how EGFR-GS coupling influences spatial patterning of checkpoint ligands and to test whether inhibition of neuraminidases enhances immune recognition in vivo.