Distribution Of Membrane Proteins Research Articles

A dynamic partitioning mechanism spatiotemporally polarizes membrane protein distributions during amoeboid migration and cortical patterning

A dynamic partitioning mechanism spatiotemporally polarizes membrane protein distributions during amoeboid migration and cortical patterning

A dynamic partitioning mechanism polarizes membrane protein distribution

The plasma membrane is widely regarded as the hub of the numerous signal transduction activities. Yet, the fundamental biophysical mechanisms that spatiotemporally compartmentalize different classes of membrane proteins remain unclear. Using multimodal live-cell imaging, here we first show that several lipid-anchored membrane proteins are consistently depleted from the membrane regions where the Ras/PI3K/Akt/F-actin network is activated. The dynamic polarization of these proteins does not depend upon the F-actin-based cytoskeletal structures, recurring shuttling between membrane and cytosol, or directed vesicular trafficking. Photoconversion microscopy and single-molecule measurements demonstrate that these lipid-anchored molecules have substantially dissimilar diffusion profiles in different regions of the membrane which enable their selective segregation. When these diffusion coefficients are incorporated into an excitable network-based stochastic reaction-diffusion model, simulations reveal that the altered affinity mediated selective partitioning is sufficient to drive familiar propagating wave patterns. Furthermore, normally uniform integral and lipid-anchored membrane proteins partition successfully when membrane domain-specific peptides are optogenetically recruited to them. We propose “dynamic partitioning” as a new mechanism that can account for large-scale compartmentalization of a wide array of lipid-anchored and integral membrane proteins during various physiological processes where membrane polarizes.

Oligomeric organization of membrane proteins from native membranes at nanoscale spatial and single-molecule resolution.

The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to understanding membrane protein biology. We report Native-nanoBleach, a total internal reflection fluorescence microscopy-based single-molecule photobleaching step analysis technique to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ~10 nm. We achieved this by capturing target membrane proteins in native nanodiscs with their proximal native membrane environment using amphipathic copolymers. We applied Native-nanoBleach to quantify the oligomerization status of structurally and functionally diverse membrane proteins, including a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under growth-factor binding and oncogenic mutations, respectively. Our data suggest that Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes under physiologically and clinically relevant conditions.

Loss of EGF receptor polarity enables homeostatic imbalance in epithelial-cell models.

The polarized distribution of membrane proteins into apical and basolateral domains provides the basis for specialized functions of epithelial tissues. The EGF receptor (EGFR) plays important roles in embryonic development, adult-epithelial tissue homeostasis, and growth and survival of many carcinomas. Typically targeted to basolateral domains, there is also considerable evidence of EGFR sorting plasticity but very limited knowledge regarding domain-specific EGFR substrates. Here we have investigated effects of selective EGFR mistargeting because of inactive-basolateral sorting signals on epithelial-cell homeostatic responses to growth-induced stress in MDCK cell models. Aberrant EGFR localization was associated with multilayer formation, anchorage-independent growth, and upregulated expression of the intermediate filament-protein vimentin characteristically seen in cells undergoing epithelial-to-mesenchymal transition. EGFRs were selectively retained following their internalization from apical membranes, and a signaling pathway involving the signaling adaptor Gab1 protein and extracellular signal-regulated kinase ERK5 had an essential role integrating multiple responses to growth-induced stress. Our studies highlight the potential importance of cellular machinery specifying EGFR polarity in epithelial pathologies associated with homeostatic imbalance.

Kinetics of Ganglioside-Rich Supported Lipid Bilayer Formation with Tracer Vesicle Fluorescence Imaging.

Gangliosides, forming a class of lipids complemented by sugar chains, influence the lateral distribution of membrane proteins or membrane-binding proteins, act as receptors for viruses and bacterial toxins, and mediate several types of cellular signaling. Gangliosides incorporated into supported lipid bilayers (SLBs) have been widely applied as a model system to examine these biological processes. In this work, we explored how ganglioside composition affects the kinetics of SLB formation using the vesicle rupturing method on a solid surface. We imaged the attachment of vesicles and the subsequent SLB formation using the time-lapse total internal reflection fluorescence microscopy technique. In the early phase, the ganglioside type and concentration influence the adsorption kinetics of vesicles and their residence/lifetime on the surface before rupturing. Our data confirm that a simultaneous rupturing of neighboring surface-adsorbed vesicles forms microscopic lipid patches on the surface and it is triggered by a critical coverage of the vesicles independent of their composition. In the SLB growth phase, lipid patches merge, forming a continuous SLB. The propagation of patch edges catalyzes the process and depends on the ganglioside type. Our pH-dependent experiments confirm that the polar/charged head groups of the gangliosides have a critical role in these steps and phases of SLB formation kinetics.

Modular DNA-Origami-Based Nanoarrays Enhance Cell Binding Affinity through the "Lock-and-Key" Interaction.

Surface proteins of cells are generally recognized through receptor-ligand interactions (RLIs) in disease diagnosis, but their nonuniform spatial distribution and higher-order structure lead to low binding affinity. Constructing nanotopologies that match the spatial distribution of membrane proteins to improve the binding affinity remains a challenge. Inspired by the multiantigen recognition of immune synapses, we developed modular DNA-origami-based nanoarrays with multivalent aptamers. By adjusting the valency and interspacing of the aptamers, we constructed specific nanotopology to match the spatial distribution of target protein clusters and avoid potential steric hindrance. We found that the nanoarrays significantly enhanced the binding affinity of target cells and synergistically recognized low-affinity antigen-specific cells. In addition, DNA nanoarrays used for the clinical detection of circulating tumor cells successfully verified their precise recognition ability and high-affinity RLIs. Such nanoarrays will further promote the potential application of DNA materials in clinical detection and even cell membrane engineering.

Inhibiting Leishmania donovani Sterol Methyltransferase to Identify Lead Compounds Using Molecular Modelling.

The recent outlook of leishmaniasis as a global public health concern coupled with the reportage of resistance and lack of efficacy of most antileishmanial drugs calls for a concerted effort to find new leads. The study combined In silico and in vitro approaches to identify novel potential synthetic small-molecule inhibitors targeting the Leishmania donovani sterol methyltransferase (LdSMT). The LdSMT enzyme in the ergosterol biosynthetic pathway is required for the parasite's membrane fluidity, distribution of membrane proteins, and control of the cell cycle. The lack of LdSMT homologue in the human host and its conserved nature among all Leishmania parasites makes it a viable target for future antileishmanial drugs. Initially, six known inhibitors of LdSMT with IC50 < 10 μM were used to generate a pharmacophore model with a score of 0.9144 using LigandScout. The validated model was used to screen a synthetic library of 95,630 compounds obtained from InterBioScreen limited. Twenty compounds with pharmacophore fit scores above 50 were docked against the modelled three-dimensional structure of LdSMT using AutoDock Vina. Consequently, nine compounds with binding energies ranging from -7.5 to -8.7 kcal/mol were identified as potential hit molecules. Three compounds comprising STOCK6S-06707, STOCK6S-84928, and STOCK6S-65920 with respective binding energies of -8.7, -8.2, and -8.0 kcal/mol, lower than 22,26-azasterol (-7.6 kcal/mol), a known LdSMT inhibitor, were selected as plausible lead molecules. Molecular dynamics simulation studies and molecular mechanics Poisson-Boltzmann surface area calculations showed that the residues Asp25 and Trp208 were critical for ligand binding. The compounds were also predicted to have antileishmanial activity with reasonable pharmacological and toxicity profiles. When the antileishmanial activity of the three hits was evaluated in vitro against the promastigotes of L. donovani, mean half-maximal inhibitory concentrations (IC50) of 21.9 ± 1.5 μM (STOCK6S-06707), 23.5 ± 1.1 μM (STOCK6S-84928), and 118.3 ± 5.8 μM (STOCK6S-65920) were obtained. Furthermore, STOCK6S-84928 and STOCK6S-65920 inhibited the growth of Trypanosoma brucei, with IC50 of 14.3 ± 2.0 μM and 18.1 ± 1.4 μM, respectively. The identified compounds could be optimised to develop potent antileishmanial therapeutic agents.

Membrane curvature sensed and induced by formin binding Protein 17.

Cell functions such as motility, differentiation, protein trafficking or signal transduction, often require some membrane reshaping events involving a change in membrane curvature and protein distribution. Defects associated with the proteins involved in membrane reshaping can cause several diseases such as centronuclear myopathy or Charcot-Marie-Tooth neuropathies. Among the proteins associated with alteration in membrane curvature, BAR (Bin/Amphiphysin/Rvs) domain proteins stand out. BAR domain proteins are crescent shaped dimeric structures that bind to lipid bilayers through electrostatic interactions. These curvature sensitive proteins are capable of inducing both local and global curvatures. However, the complexity of the mechanisms of the interactions between these proteins and lipid bilayers are not well known. Computational studies of these protein-membrane interactions using molecular dynamics are often coarse-grained as opposed to all-atoms simulations. In the present study, we explore the curvature sensitivity and inducing properties of Formin Binding Protein 17 (fbp17) through all-atoms molecular dynamics simulations. We simulated fbp17 with different lipid bilayer compositions on flat and buckled bilayers. We were able to isolate the residues involved in the interactions. Our results suggest that fbp17 can induce a certain range of local curvature on various flat membranes. Fp17 can also sense or conform to various ranges of curved bilayers. These results give an insight into the role of BAR domain protein in general, and fbp17 in particular in the context of membrane structural fluctuations.

Oligomeric distribution of membrane proteins in native-membrane environment at nanoscale spatial and single-molecule resolution.

Membrane proteins comprise ∼1/4 the human proteome and play indispensable roles in cellular physiology. Recent studies have highlighted the role of oligomerization and clustering in membrane protein-mediated signaling and physiological responses. The protein-centricity of experimental biophysics has led to a view of membrane protein function devoid of two-dimensional cell membranes, which play pivotal roles in their regulation, structure, oligomerization, and function. Quantitation of membrane protein oligomeric states directly from native-membrane environments remains challenging. Some of the main problems are: an inability to preserve the native-membrane environment while attaining precise spatial and high molecular resolution, insufficient sensitivity to detect and analyze membrane proteins at endogenous levels of expression, and dependence on bulk analyses that lack single-molecule information. We propose a single-molecule total internal reflection fluorescence microscopy (TIRF)-based imaging assay to quantify membrane protein oligomeric states directly from native-membrane environments at nanoscale spatial (∼10 nm) and single-molecule resolution. We benchmarked and validated this experimental platform using several membrane proteins with well-established oligomeric states. We then applied this method to quantify as yet unknown oligomeric distributions of diverse membrane-proteins from E. Coli, mammalian cells, and patient-derived cell lines expressing target proteins at endogenous levels. We combined this innovative experimental approach with mutagenesis, truncations, antibody/nanobody binding, and inhibitor/drug treatments to (1) identify changes in oligomeric states and (2) map oligomeric interfaces in membrane proteins. We finally correlated our results with membrane-localized signaling to connect the physical modulation of protein oligomeric states to physiological outcomes. Our approach can be extended to identify compounds/antibodies that can modulate oligomeric distributions to ablate/alter signaling in disease-relevant membrane proteins. This general experimental pipeline ushers in a new era of studying membrane proteins in their native-membrane environments at an unprecedented spatial and molecular resolution.

Oligomerization of peripheral membrane proteins provides tunable control of cell surface polarity

Asymmetric distributions of peripheral membrane proteins define cell polarity across all kingdoms of life. Non-linear positive feedback on membrane binding is essential to amplify and stabilize these asymmetries, but how specific molecular sources of non-linearity shape polarization dynamics remains poorly understood. Here we show that the ability to oligomerize, which is common to many peripheral membrane proteins, can play a profound role in shaping polarization dynamics in simple feedback circuits. We show that size-dependent binding avidity and mobility of membrane-bound oligomers endow polarity circuits with several key properties. Size-dependent membrane binding avidity confers a form of positive feedback on the accumulation of oligomer subunits. Although insufficient by itself, this sharply reduces the amount of additional feedback required for spontaneous emergence and stable maintenance of polarized states. Size-dependent oligomer mobility makes symmetry breaking and stable polarity more robust with respect to variation in subunit diffusivities and cell sizes, and slows the approach to a final stable spatial distribution, allowing cells to “remember” polarity boundaries imposed by transient external cues. Together, these findings reveal how oligomerization of peripheral membrane proteins can provide powerful and highly tunable sources of non-linear feedback in biochemical circuits that govern cell surface polarity. Given its prevalence and widespread involvement in cell polarity, we speculate that self-oligomerization may have provided an accessible path to evolving simple polarity circuits.

On-grid labeling method for freeze-fracture replicas.

Sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) is an electron microscopic (EM) method that can define the two-dimensional distribution of membrane proteins and lipids in a quantitative manner. Despite its unsurpassed merit, SDS-FRL has been adopted in a limited number of labs, probably because it requires a laborious labeling process as well as equipment and technique for freeze-fracture. Here, we present a method that reduces the manual labor significantly by mounting freeze-fracture replicas on EM grids prior to labeling. This was made possible by the discovery that freeze-fracture replicas invariably adhere to the carbon-coated formvar membrane with their platinum-carbon side, ensuring that the membrane molecules retained in replicas are accessible to labeling solutions. The replicas mounted on EM grids can be stored dry until labeling, checked by light microscopy before labeling and labeled in the same manner as tissue sections. This on-grid method will make SDS-FRL easier to access for many researchers.

Freeze-Fracture Electron Microscopy for Extracellular Vesicle Analysis.

Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs' diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.

Correlated STORM-homoFRET imaging reveals highly heterogeneous membrane receptor structures

Mapping the self-organization and spatial distribution of membrane proteins is key to understanding their function. Developing methods that can provide insight into correlations between membrane protein colocalization and interactions is challenging. We report here on a correlated stochastic optical reconstruction microscopy/homoFRET imaging approach for resolving the nanoscale distribution and oligomeric state of membrane proteins. Using live cell homoFRET imaging of carcinoembryonic antigen-related cellular adhesion molecule 1, a cell-surface receptor known to exist in a complex equilibrium between monomer and dimer/oligomer states, we revealed highly heterogeneous diffraction-limited structures on the surface of HeLa cells. Furthermore, correlated super-resolved stochastic optical reconstruction microscopy imaging showed that these structures comprised a complex mixture and spatial distribution of self-associated carcinoembryonic antigen-related cellular adhesion molecule 1 molecules. In conclusion, this correlated approach provides a compelling strategy for addressing challenging questions about the interplay between membrane protein concentration, distribution, interaction, clustering, and function.

Ubiquitin-specific protease TRE17/USP6 promotes tumor cell invasion through the regulation of glycoprotein CD147 intracellular trafficking

Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases, the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances matrix metalloproteinase synthesis and matrix degradation, thereby promoting tumor cell invasion.

Rho and Rab Family Small GTPases in the Regulation of Membrane Polarity in Epithelial Cells.

Membrane polarity, defined as the asymmetric distribution of lipids and proteins in the plasma membrane, is a critical prerequisite for the development of multicellular tissues, such as epithelia and endothelia. Membrane polarity is regulated by polarized trafficking of membrane components to specific membrane domains and requires the presence of intramembrane diffusion barriers that prevent the intermixing of asymmetrically distributed membrane components. This intramembrane diffusion barrier is localized at the tight junctions (TJs) in these cells. Both the formation of cell-cell junctions and the polarized traffic of membrane proteins and lipids are regulated by Rho and Rab family small GTPases. In this review article, we will summarize the recent developments in the regulation of apico-basal membrane polarity by polarized membrane traffic and the formation of the intramembrane diffusion barrier in epithelial cells with a particular focus on the role of Rho and Rab family small GTPases.

Pseudocleavage furrows restrict plasma membrane-associated PH domain in syncytial Drosophila embryos

Syncytial cells contain multiple nuclei and have local distribution and function of cellular components despite being synthesized in a common cytoplasm. The syncytial Drosophila blastoderm embryo shows reduced spread of organelle and plasma membrane-associated proteins between adjacent nucleo-cytoplasmic domains. Anchoring to the cytoarchitecture within a nucleo-cytoplasmic domain is likely to decrease the spread of molecules; however, its role in restricting this spread has not been assessed. In order to analyze the cellular mechanisms that regulate the rate of spread of plasma membrane-associated molecules in the syncytial Drosophila embryos, we express a pleckstrin homology (PH) domain in a localized manner at the anterior of the embryo by tagging it with the bicoid mRNA localization signal. Anteriorly expressed PH domain forms an exponential gradient in the anteroposterior axis with a longer length scale compared with Bicoid. Using a combination of experiments and theoretical modeling, we find that the characteristic distribution and length scale emerge due to plasma membrane sequestration and restriction within an energid. Loss of plasma membrane remodeling to form pseudocleavage furrows shows an enhanced spread of PH domain but not Bicoid. Modeling analysis suggests that the enhanced spread of the PH domain occurs due to the increased spread of the cytoplasmic population of the PH domain in pseudocleavage furrow mutants. Our analysis of cytoarchitecture interaction in regulating plasma membrane protein distribution and constraining its spread has implications on the mechanisms of spread of various molecules, such as morphogens in syncytial cells.

Gold In-and-Out: A Toolkit for Analyzing Subcellular Distribution of Immunogold-Labeled Membrane Proteins in Freeze-Fracture Replica Images.

Integral membrane proteins such as ion channels, transporters, and receptors shape cell activity and mediate cell-to-cell communication in the brain. The distribution, quantity, and clustering arrangement of those proteins contribute to the physiological properties of the cell; therefore, precise quantification of their state can be used to gain insight into cellular function. Using a highly sensitive immunoelectron microscopy technique called sodium dodecyl sulfate-digested freeze-fracture replica immunogold labeling (SDS-FRL), multiple membrane proteins can be tagged with different sizes of immunogold particles at once and visualized two-dimensionally. For quantification, gold particles in the images must be annotated, and then different mathematical and statistical methods must be applied to characterize the distribution states of proteins of interest. To perform such analyses in a user-friendly manner, we developed a program with a simple graphical user interface called Gold In-and-Out (GIO), which integrates several classical and novel analysis methods for immunogold labeled replicas into one self-contained package. GIO takes an input of particle coordinates, then allows users to implement analysis methods such as nearest neighbor distance (NND) and particle clustering. The program not only performs the selected analysis but also automatically compares the results of the real distribution to a random distribution of the same number of particles on the membrane region of interest. In addition to classical approaches for analyzing protein distribution, GIO includes new tools to analyze the positional bias of a target protein relative to a morphological landmark such as dendritic spines, and can also be applied for synaptic protein analysis. Gold Rippler provides a normalized metric of particle density that is resistant to differences in labeling efficiency among samples, while Gold Star is useful for quantifying distances between a protein and landmark. This package aims to help standardize analysis methods for subcellular and synaptic protein localization with a user-friendly interface while increasing the efficiency of these time-consuming analyses.

A particle size threshold governs diffusion and segregation of PAR-3 during cell polarization.

SUMMARYThe actomyosin cortex regulates the localization and function of proteins at the plasma membrane. Here, we study how membrane binding, cortical movements, and diffusion determine membrane protein distribution. In Caenorhabditis elegans zygotes, actomyosin flows transport PAR polarity proteins to establish the anterior-posterior axis. Oligomerization of a key scaffold protein, PAR-3, is required for polarization. PAR-3 oligomers are a heterogeneous population of many different sizes, and it remains unclear how oligomer size affects PAR-3 segregation. To address this question, we engineered PAR-3 to defined sizes. We report that PAR-3 trimers are necessary and sufficient for PAR-3 function during polarization and later embryo development. Quantitative analysis of PAR-3 diffusion shows that a threshold size of three subunits allows PAR-3 clusters to stably bind the membrane, where they are corralled and transported by the actomyosin cortex. Our study provides a quantitative model for size-dependent protein transportation of peripheral membrane proteins by cortical flow.

Spatial regulation of endosomes in growing dendrites

Many membrane proteins are highly enriched in either dendrites or axons. This non-uniform distribution is a critical feature of neuronal polarity and underlies neuronal function. The molecular mechanisms responsible for polarized distribution of membrane proteins has been studied for some time and many answers have emerged. A less well studied feature of neurons is that organelles are also frequently non-uniformly distributed. For instance, EEA1-positive early endosomes are somatodendritic whereas synaptic vesicles are axonal. In addition, some organelles are present in both axons and dendrites, but not distributed uniformly along the processes. One well known example are lysosomes which are abundant in the soma and proximal dendrite, but sparse in the distal dendrite and the distal axon. The mechanisms that determine the spatial distribution of organelles along dendrites are only starting to be studied. In this review, we will discuss the cell biological mechanisms of how the distribution of diverse sets of endosomes along the proximal-distal axis of dendrites might be regulated. In particular, we will focus on the regulation of bulk homeostatic mechanisms as opposed to local regulation. We posit that immature dendrites regulate organelle motility differently from mature dendrites in order to spatially organize dendrite growth, branching and sculpting.

Neonatal hypoxia ischemia redistributes L1 cell adhesion molecule into rat cerebellar lipid rafts.

Hypoxic-ischemic encephalopathy (HIE) is a devastating disease with lifelong disabilities. Hypothermia is currently the only treatment. At term, the neonatal cerebellum may be particularly vulnerable to the effects of HIE. At this time, many developmental processes depend on lipid raft function. These microdomains of the plasma membrane are critical for cellular signaling and axon extension. We hypothesized that HIE alters the protein content of lipid rafts in the cerebellum. Postnatal day (PN) 10 animals, considered human term equivalent, underwent hypoxic-ischemic (HI) injury by a right carotid artery ligation followed by hypoxia. For some animals, LPS was administered on PN7, and hypothermia (HT) was conducted for 4 h post-hypoxia. Lipid rafts were isolated from the right and left cerebella. The percent of total L1 cell adhesion molecule in lipid rafts was determined 4 and 72 h after hypoxia. No sex differences were found. HI alone caused significant increases in the percent of L1 in lipid rafts which persisted until 72 h in the right but not the left cerebellum. A small but significant effect of LPS was detected in the left cerebellum 72 h after HI. Hypothermia had no effect. Lipid rafts may be a new target for interventions of HIE. This article investigates the effect of neonatal exposure to hypoxic-ischemic encephalopathy (HIE) on the distribution of membrane proteins in the cerebellum. This article explores the effectiveness of hypothermia as a prevention for the harmful effects of HIE on membrane protein distribution. This article shows an area of potential detriment secondary to HIE that persists with current treatments, and explores ideas for new treatments.