Biology subjects

Waxham, M. N.

Publications and source records attributed to Waxham, M. N..

9 recordsLinked to original sources

bioRxiv2026

CaMKII generates actin bundle morphology and mechanics distinct from canonical bivalent cross-linkers

Actin cross-linkers are essential modulators of the actin cytoskeleton, enabling structural diversity and dynamic remodeling. Among them, calcium/calmodulin-dependent protein kinase II (CaMKII) is unique in serving a dual role as a kinase and as a multivalent structural binder of actin in dendritic spines, where it plays a major role in supporting dendritic spine structure via its {beta} subunit. To understand the basis of the structural function of CaMKII, we quantified the morphology and mechanics of actin bundles formed by CaMKII and directly compared them with bundles assembled by the canonical bivalent cross-linkers -actinin and fascin. Using fluorescence microscopy, we measured contour length, straightness ratio, and persistence length across different cross-linker concentrations. Fascin generated progressively shorter, straighter, and stiffer bundles with increasing concentration, whereas -actinin bundles remained largely unchanged. In contrast, increasing CaMKII concentration reduced both bundle straightness and persistence length, revealing a unique concentration-dependent increase in bundle flexibility. To investigate the structural origins of these mechanical behaviors, we complemented the experiments with coarse-grained simulations of cross-linked actin bundles. Simulations revealed that CaMKII-generated bundles retained a substantially larger fraction of their curvature in their time-averaged configuration than bundles formed by -actinin or fascin, indicating that bundle mechanics are strongly influenced by organizational features beyond thermal bending fluctuations alone. CaMKII bundles displayed behavior consistent with additional structural complexity arising from their multivalent architecture and flexible linker domains. Together, our experimental and computational results identify CaMKII as a structurally and mechanically distinct actin cross-linker. We propose that the combination of multivalency and linker flexibility enables CaMKII to function as a molecular structural pivot, promoting flexible, adaptable actin assemblies that are mechanically compatible with the dynamic remodeling required for dendritic spine plasticity.

biophysics↗

bioRxiv2026

Spatial confinement shapes organelle architecture and remodeling in axons

Axons are extended cellular compartments that mediate neuronal connectivity over extraordinary distances, placing unique demands on local organelle organization and trafficking. How these geometric constraints influence organelle architecture remains poorly understood. Here, we used cryo-electron tomography and quantitative morphometric analyses to define the three-dimensional ultrastructural organization of dorsal root ganglion axons in a near-native state. We identify distinct vesicle populations with tightly versus broadly constrained size distributions and reveal a continuum of endolysosomal and autophagic intermediates that highlight the dynamic nature of membrane remodeling in growing axons. Unexpectedly, we observe vesicular structures enclosed within the lumen of the endoplasmic reticulum, suggesting a previously undescribed mechanism of ER membrane remodeling. Across multiple organelle classes, morphology and size are constrained by axonal geometry. This principle is most evident in mitochondria, which undergo dramatic narrowing and remodeling at varicosity - axon boundaries to traverse confined axonal segments. Together, these findings reveal spatial confinement as a fundamental organizing principle of axonal cell biology. SUMMARYCryo-electron tomography establishes a quantitative framework for organelle organization in axons. Diverse membrane trafficking pathways, including endolysosomal intermediates and mitochondria, exhibit structural adaptations to axonal geometry, identifying spatial confinement as a fundamental organizing principle of axonal cell biology.

neuroscience↗

bioRxiv2025

Nanodomain formation in lipid bilayers II: The influence of mixed-chain saturated lipids

An important class of lipids found in biological membranes are composed of two structurally different hydrocarbon chains. Among these, low-melting lipids possessing both a saturated and unsaturated chain have been intensely studied because of their biological abundance and influence on lipid rafts. In contrast, much less is known about the biophysical effects of mixed chains in high-melting lipids. Here, we investigated two such lipids--MSPC (14:0-18:0 PC) and SMPC (18:0-14:0 PC)--to determine how chain length mismatch and acyl chain position on the glycerol backbone influence lateral organization. We studied the temperature-and composition-dependent phase behavior of liposomes composed of either mixed-chain or symmetric-chain high-melting lipids plus DOPC and cholesterol, using techniques sensitive to domain formation at both microscopic and nanoscopic length scales. All studied mixtures exhibited liquid-ordered (Lo) + liquid-disordered (Ld) phase coexistence with domains that were visible in confocal microscopy experiments. FRET measurements showed that all mixtures also exhibited nanoscopic heterogeneity at temperatures above the microscopic miscibility transition temperature, and cryo-EM imaging further revealed bilayer thickness variation consistent with coexisting Ld and Lo phases. Both the microscopic miscibility transition temperature, m-Tmix, and its nanoscopic counterpart, nm-Tmix, were strongly correlated with the melting transition temperature of the saturated lipid; the sole exception was SMPC/DOPC/Chol, whose m-Tmix showed a significant negative deviation from the expected value, implying an enhanced propensity for nanoscopic phase separation in mixtures containing this high-melting species. These results point to strong effects of acyl chain position within mixed-chain high-TM lipids on the microscopic phase behavior of ternary mixtures.

biophysics↗

bioRxiv2025

Primary cortical neurons form calcium-phosphate sheets with a bone-precursor-like ultrastructure

Calcium-phosphate (CaP) is a ubiquitous inorganic compound that plays an important structural role in healthy bone and teeth formation, but its pathologic buildup can occur in dyshomeostatic calcium disorders like Alzheimers disease and Leigh syndrome. The nexus of pathologic extracellular CaP in the nervous system is not well understood, but prior evidence suggests mitochondria could be a source. We have observed mitochondria-sized sheet-like CaP aggregates within functional wild type cortical neuron cultures at 1 and 20 DIV. Neurons were extracted from embryonic day 18 (E18) rat embryos following standard protocols to study neuronal structure and function. We have used a combination of cryo-ET, cryo-CLEM, and LDSAED to demonstrate that these aggregates are octacalcium phosphate-like, are associated with mitochondria, and that at least a portion are extruded via migrasomes. Visually similar aggregates were previously observed in Huntingtons disease model neurons, but in that study they were not observed in WT controls. These findings show that this CaP aggregation process occurs routinely in WT neurons and may reveal an important link for how mitochondria may participate in calcification, highlighting them as potential therapeutic targets in neurological disorders characterized by pathological calcification, such as Alzheimers disease.

neuroscience↗

bioRxiv2023

Role of a Pdlim5:PalmD complex in directing dendrite morphology

Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching (Baumert et al., J Cell Biol 2020). We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., via adducin/spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that each proteins effects are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.

neuroscience↗

bioRxiv2022

Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers

Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. SignificanceRaft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.

biophysics↗

bioRxiv2019

On the mechanism of bilayer separation by extrusion; or, why your large unilamellar vesicles are not really unilamellar

Extrusion through porous filters is a widely used method for preparing biomimetic model membranes. Of primary importance in this approach is the efficient production of single bilayer (unilamellar) vesicles that eliminate the influence of interlamellar interactions and strictly define the bilayer surface area available to external reagents such as proteins. Sub-microscopic vesicles produced using extrusion are widely assumed to be unilamellar, and large deviations from this assumption would dramatically impact interpretations from many model membrane experiments. Using three probe-free methods--small-angle X-ray and neutron scattering (SAXS and SANS) and cryogenic electron microscopy (cryoEM)--we report unambiguous evidence of extensive multilamellarity in extruded vesicles composed of neutral phosphatidylcholine lipids, including for the common case of neutral lipids dispersed in physiological buffer and extruded through 100 nm diameter pores. In such preparations, only ~35% of lipids are externally accessible, and this fraction is highly dependent on preparation conditions. Charged lipids promote unilamellarity, as does decreasing solvent ionic strength, indicating the importance of electrostatic interactions in determining the lamellarity of extruded vesicles. Smaller extrusion pore sizes also robustly increase the fraction of unilamellar vesicles, suggesting a role for membrane bending. Taken together, these observations suggest a mechanistic model for extrusion, wherein formation of unilamellar vesicles involves competition between bilayer bending and adhesion energies. The findings presented here have wide-ranging implications for the design and interpretation of model membrane studies, especially ensemble-averaged observations relying on the assumption of unilamellarity.\n\nSTATEMENT OF SIGNIFICANCEExtruded vesicles are a ubiquitous tool in membrane research. It is widely presumed that extrusion produces unilamellar (i.e., single bilayer) vesicles, an assumption that is often crucial for data analysis and interpretation. Using X-ray and neutron scattering and cryogenic electron microscopy, we show that a substantial amount of lipid remains inaccessible after extrusion due to an abundance of multilamellar vesicles (MLVs). While this is a general phenomenon for neutral lipids, MLV contamination can be reduced by several complementary approaches such as including charged lipids in the mixture, reducing the ionic strength of the aqueous medium, and reducing the extrusion pore size. These observations together suggest a mechanism by which extrusion strips MLVs of their layers.

biophysics↗

bioRxiv2018

Visualization of the type III secretion mediated Salmonella-host cell interface using cryo-electron tomography

Many important gram-negative bacterial pathogens use highly sophisticated type III secretion systems (T3SSs) to establish complex host-pathogen interactions. Bacterial-host cell contact triggers the activation of the T3SS and the subsequent insertion of a translocon pore into the target cell membrane, which serves as a conduit for the passage of effector proteins. Therefore the initial interaction between T3SS-bearing bacteria and host cells is the critical step in the deployment of the protein secretion machine, yet this process remains poorly understood. Here, we use high-throughput cryo-electron tomography (cryo-ET) to visualize the T3SS-mediated Salmonella-host cell interface. Our analysis reveals the intact translocon at an unprecedented level of resolution, its deployment in the host cell membrane, and the establishment of an intimate association between the bacteria and the target cells, which is essential for effector translocation. Our studies provide critical data supporting the long postulated direct injection model for effector translocation.

microbiology↗

bioRxiv2018

Morphological Adaptations of Mitochondria in Spatially Restricted Axons Revealed by Cryo-Electron Tomography

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphological characteristics of mitochondria residing at the narrowed boundary between presynaptic varicosities and axons with limited physical dimensions (~80 nm), compared to mitochondria in non-spatially restricted environments. Furthermore, segmentation of the tomograms revealed discrete organizations between the inner and outer membranes, suggesting possible independent remodeling of each membrane in mitochondria at spatially restricted axonal/varicosity boundaries. Thus, cryo-ET of mitochondria within axonal subcompartments reveals that spatial restrictions do not obstruct mitochondria from residing within them but limited available space can influence their gross morphology and the organization of the inner and outer membranes. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlights the potential for remarkable structural plasticity of mitochondria to adapt to spatial restrictions within axons.

neuroscience↗