MD Arthur James
email: arthur.james@aphp.fr

MD Youri Yordanov

PhD Sylvain Ausset

MD Matthieu Langlois

MD Jean-Pierre Tourtier

MD Pierre Carli

MD Bruno Riou

PhD Mathieu Raux

Prof M James

Langlois

Assessment of the Mass Casualty Triage during the November 2015 Paris Area Terrorist Attacks: Towards a Simple Triage Rule

Keywords: Triage, Disaster medicine, Emergency Surgery, Gunshot wound, Mass casualty, Terrorism

Key Points

(words count: 109) Question: Was the prehospital triage performed during the 2015 Paris terrorist attack appropriate ?

Findings: Among 337 casualties admitted to the hospital, 262 (78%) were triaged, 74 (22%) as absolute emergency (AE), 188 (56%) as relative emergency (RE). The proportion of appropriately classified was 0.82 [95%CI 0.76-0.86], undertriage 0.36 [95%CI 0.27-0.47], and overtriage 0.08 [95%CI 0.04-0.13], resulting in no detectable adverse effect when considering mortality. The qualitative analysis of severe under-and overtriage indicates some room for improvement.

Meaning: A simple binary triage categorization can be performed on scene in a large proportion of casualties and had good diagnostic performance during a massive event with multisite and multitype attack.

Abstract (263 words)

BACKROUND : Triage is a key principle in the management of mass casualty incidents. The objective of this study was to assess the triage performed during the 2015 Paris area terrorist attack.

METHODS : This was a retrospective cohort study collecting medical data from all casualties of the attacks on November 13 th 2015 in Paris and its suburb Seine Saint-Denis, France, with body injuries , who arrived alive at any hospital within the first 24 hours after the events.

Prehospital triage was performed using a dichotomous scale of absolute emergencies (AE) and relative emergencies (RE), which was then compared to that performed by an expert panel (reference method). FINDINGS: Among, 337 casualties admitted to the hospital, 262 (78%) were categorized during the prehospital phase, 74 (22%) as AE, 188 (56%) as RE. The expert categorization was AE in 119 (35%) and RE in 218 (65%) of cases. Undertriage was 0.36 [95%CI 0.27-0.47], overtriage 0.08 [95%CI 0.04-0.13], and the proportion of appropriately classified 0.82 [95%CI 0.76-0.86]. A subgroup of casualties (n=115) were triaged again at arrival to the hospital, but the diagnostic performance was not significantly modified. Among undertriaged casualties, 8 (23%) were considered as being severely undertriaged. Among overtriaged casualties, 10 (77%) were considered as being severely overtriaged. The observed mortality in the whole cohort (n=7; 2.1%) was not significantly different from that expected (n=11, 3.3%; P=0×92). INTERPRETATION: A simple triage categorization had good diagnostic performance, resulting in no detectable adverse effect when considering mortality. Qualitative analysis of under-and overtriage indicate some possibilities for further improvement.

FUNDING : Assistance Publique-Hôpitaux de Paris

Introduction

Major incidents resulting in mass casualties occurs more frequently and challenge our ability to provide the best care to most people. Unfortunately, western countries have experienced an increasing number terrorist attacks using fire-arms, explosives, and trucks. [START_REF] Aylwin | Reduction in critical mortality in urban mass casualty incidents: analysis of triage, surge, and resource use after the London bombings on July 7[END_REF][START_REF] Hirsch | The medical response to multisite terrorist attacks in Paris[END_REF][START_REF] Carles | Mass casualty events and health organization: terrorist attack in Nice[END_REF][START_REF] Carli | The French emergency medical services after the Paris and Nice terrorist attacks: what have we learnt?[END_REF][START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] These attacks designed to kill and injure the largest number of casualties, using simultaneous attacks and/or multiple means, require an appropriate medical organization. [START_REF] Hirsch | The medical response to multisite terrorist attacks in Paris[END_REF] Triage is a key principle in the management of major incidents, comprising early identification and transport to the most appropriate center within the most appropriate delay. Although both undertriage and overtriage are of paramount importance during a major incident, there is limited evidence based on existing triage tools. Since prospective research in this area is difficult to conduct, and probably unethical, most information has been obtained during simulation research which may not apply to real conditions. [START_REF] Horne | Triage in the defense medical services[END_REF][START_REF] Sacco | Precise formulation and evidence-based application of resource-constrained triage[END_REF][START_REF] Lee | Impact of a two-step emergency department triage model with START, then CTAS, on patient flow during a simulated mass-casualty incident[END_REF] Analysis of the triage during major incidents may also be difficult because of either small sample size, study heterogeneity, and/or biased selection of studied casualties. [START_REF] Turner | Prehospital management of mass casualty civilian shootings: a systematic literature review[END_REF][START_REF] Pinkert | Primary triage, evacuation priorities, and rapid primary distribution between adjacent hospitals. Lessons learned from a suicide bomber attack in downtown Tel-Aviv[END_REF] A systematic review concluded that field triage systems do not perform consistently during mass casualty events. [START_REF] Timbie | Systematic review of strategies to manage and allocate scarce resources during mass casualty events[END_REF] In 2015, Paris and its suburb Seine Saint-Denis were the scene of multiple terrorist attacks which were unprecedented since the Second World War in Europe. [START_REF] Carles | Mass casualty events and health organization: terrorist attack in Nice[END_REF][START_REF] Carli | The French emergency medical services after the Paris and Nice terrorist attacks: what have we learnt?[END_REF][START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] In the present study, we analyze the prehospital triage which was performed using a dichotomous scale (absolute emergencies (AE) vs relative emergencies (RE)). This simple triage tool was chosen because it corresponded to that taught and practiced in France for more than 30 years now, [START_REF] Carli | Terrorism in France[END_REF] following the principle of simplicity recommended after the London attacks in 2005. [START_REF] Aylwin | Reduction in critical mortality in urban mass casualty incidents: analysis of triage, surge, and resource use after the London bombings on July 7[END_REF] We aimed at assessing the diagnostic performance of this triage in this unique cohort of patients, on a quantitative and qualitative basis, and test its reliability. We believe this analysis is important and provides relevant information to improve our emergency plans.

Material and methods

This observational retrospective cohort study used medical data from casualties of the Paris area terrorist attacks, collected anonymously, as recently described. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] Data processing authorization was obtained from the Commission Nationale Informatique et Liberté and the Comité de Protection des Personnes Ile de France, exempting casualties from giving their consent for the use of their data for observational research purposes. This report follows the STROBE recommendations. [START_REF] Von Elm | The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies[END_REF]

Study population

Casualties included in the study were those with somatic lesions and who arrived alive at the hospital within the first 24h after the attacks of 13 November 2015. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] Casualties without somatic lesions, consulting for psychological trauma, those with no recorded medical condition and who did not require hospitalization, or those presenting to hospital later after the events were excluded. Casualties were managed and transported to hospitals by mobile intensive care units and/or by Fire-Brigade ambulances according to the French standards of prehospital care. [START_REF] Adnet | International EMS systems: France[END_REF] Hospital orientation was managed by the Service d'Aide Médicale Urgente (SAMU) a medicalized civil emergency medical service based on the pre-hospital evaluation and categorization. When needed, priority was given to secure the premises before access to casualties and their evacuation to hospitals in the Paris region, five of them being civilian level-1 trauma centers. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] The two military hospitals were also considered as level-1 trauma centers.

Patients who were declared « dead on scene » (decision taken by an emergency physician) were not taken by the EMS but by police units and thus not transported to an hospital but directly to the legal medicine institute which is centralized for the Paris area and not situated in an hospital.

Measurement and analysis

Casualties were identified thought police and hospital registers. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] Descriptive data, wounding process, anatomical lesions, pre-hospital triage, mean times of transfer to hospital and surgery rooms, and therapeutic and diagnostic measures were collected using each casualty medical files. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] The revised trauma score (RTS) scores, Abbreviated Injury Score (AIS), Injury Severity Score (ISS), and the Trauma Related Injury Severity Score (TRISS) were calculated. [START_REF] Boyd | Evaluating trauma care: the TRISS method. Trauma Score and the Injury Severity Score[END_REF][START_REF] Riou | The distribution of the probability of survival is a strategic issue in randomized trial in trauma[END_REF] Observed mortality was defined as the occurrence of death during hospitalization and compared to expected mortality. [START_REF] Riou | The distribution of the probability of survival is a strategic issue in randomized trial in trauma[END_REF] Transfer was defined as referring the casualty to a hospital other than the one that initially received the casualty, within 24 hours of the injury process. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] Casualties were categorized as AE or RE (Electronic supplement Table S1) then referred to level-1, 2 or 3 trauma centers with appropriate level of care. The pre-hospital categorization came from administrative files of the Préfecture de Police de Paris and from the Assistance Public-Hôpitaux de Paris respectively named "SINUS" and "VICTIMS. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] In a subgroup of casualties admitted into the level 1 trauma centers, a secondary triage was performed at admission to the hospital (AE/RE) by experienced senior physicians. The final categorization (reference method) was established independently by two senior experts (AJ, MR) from an examination of the complete medical chart (Kappa score 0.92). [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] In case of disagreement a consensus was reached by a third expert (BR).

To qualitatively assess the performance of triage, we analyzed undertriage (those classified RE in the prehospital phase but AE by the experts) and overtriage (those classified AE in the prehospital phase but RE by the experts) of the prehospital triage process. Among undertriaged casualties, we selected those with at least one anatomic lesion with AIS scoring > 3 (i.e., severe undertriage). Conversely, among overtriaged casualties, we selected those with only anatomical lesions AIS scoring 1 or 2 (i.e., severe overtriage). In both undertriaged and overtriaged casualties, those with AIS scoring = 3 were considered as equivocal (i.e., non severe under/overtriage).

Lastly, we compared prehospital triage performed in real condition to simulated triage using the three most widely used triage tools: the Field Triage Score (FTS), [START_REF] Eastridge | Field triage score (FTS) in battlefield casualties: validation of a novel triage technique in a combat environment[END_REF] the Simple Triage and Rapid Treatment (START) algorithm, [START_REF] Super | START: Simple triage and rapid treatment plan[END_REF] and the more recent Modified Physiological Triage Tool (MPTT). [START_REF] Vassallo | Major incident triage: Derivation and comparative analysis of the Modified Physiological Triage Tool (MPTT)[END_REF] Since these triage tools were based on a 3 levels classification, we transformed them into dichotomous tools as follows: FTS 0 or 1, START "immediate" and MPTT "P1" were considered as AE, whereas FTS 2, START "minor" and "delayed", and MPTT "P2" and "P3" were considered as RE. These simulated triages were established independently by two senior experts (JPT, AJ) who accessed only to variables available in prehospital conditions. This analysis took place one year after the reference categorization without knowledge of it. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] In case of disagreement a consensus was reached by a third expert (MR). Kappa scores for TS, START, and MPTT were 0.95, 0.92, and 0.50 respectively. To compare these scores to prehospital triage, the main criteria was the proportion of patients appropriately classified.

Statistical analysis

Qualitative variables are presented by number and percentage. Quantitative variables are presented by their mean+standard deviation or median [interquartile] according to the normality of the distribution. Comparison between groups was performed using the Fisher exact method, the Student t test, and the Mann Whitney test. To assess the diagnostic performance, we calculated the undertriage (1-sensitivity), overtriage (1-specificity), negative and positive predictive values, negative and positive likelihood ratio, and proportion of patients appropriately classified (Electronic supplement S2). Comparison of undertriage, overtriage and proportion of appropriately classified was performed using the Mc Nemar test. Comparisons of predictive values and likelihood ratios were performed. [START_REF] Moskowitz | Comparing the predictive values of diagnostic tests: sample size and analysis for paired study designs[END_REF][START_REF] Gu | Estimating the capacity for improvement in risk prediction with a marker[END_REF] The Bonferroni correction was applied for multiple comparisons. Missing data were not replaced. We used Kappa statistic to measure inter-rater reliability when several experts was involved to categorize a single parameter. A Kappa close to 0 indicates no agreement when a Kappa close to 1 indicates a perfect agreement.

All comparisons were two-tailed and a P value <0×05 was considered significant. Statistical analyses were carried out using R (version 3.6.1) software.

Results

Among 543 casualties with body injuries, 337 were admitted to emergency services/trauma centers (Figure 1). Two hundred and sixty two (68%) of these casualties were categorized during the prehospital phase, 74 (28%) as AE, 188 (72%) as RE. One hundred and fifteen (34%) casualties were again categorized at admission into level 1 trauma centers, 52 (46%) as AE, 63 (64%) as RE. The expert hospital categorization was AE in 119 (35%) and RE in 218 (65%) of cases (Table 1, Figure 2). In casualties not triaged during the prehospital phase (n=75), there were 23 (31%) AE and 52 (69%) RE. Secondary transfer occurred in 27 (8%) casualties ( 12AE and 15 RE), 4 of them being referred to a level-1 trauma center (1 AE and 3 RE).

The diagnostic performance of prehospital triage is shown in Table 2. The comparison of prehospital and hospital triages is shown in table 3. Among undertriaged casualties (n=35), 8 (23%) were considered as severely undertriaged. Among overtriaged casualties (n=13), 10 (77%) were considered as severely overtriaged. The main causes of severe undertriage were related to thoraco-abdominal and head penetrating injuries and most severe cause of overtriage were related to superficial limb lesions (Electronic supplement Table S3).

Using simulated triage with access only to prehospital variables, the diagnostic performance of FTS, START, and MPTT were significantly lower than that observed in our cohort when considering the proportion of casualties appropriately classified. Although the overtriage of FTS, START, and MPTT was worse than that observed in our cohort, undertriage in this group was better (Electronic supplement Table S4).

The observed in-hospital mortality (n=7; 2×1%) was not significantly different from that expected (n=11; 3.3%; P=0×92).

Discussion

Synthesis

Our study shows that a simple triage categorization (AE/RE) was performed on scene in a large proportion of casualties (78%) and had good diagnostic performance during highly complex incident involving multiple incident sites, and both gunshot and explosion. This diagnostic performance did not result in detectable adverse effects since the observed mortality during this mass casualty incident was not higher than that expected during routine trauma care.

Nevertheless, the qualitative analysis of severe under-and overtriage indicates that there is some room for improvement. In a subgroup of casualties, we observed that secondary triage at hospital admission did not significantly improve the proportion of patients appropriately classified. Lastly, simulated triage using other triage tools (FTS, START, MPTT) did not perform better than the simple prehospital triage used.

Perspectives

Wartime triage was initially meant to establish surgical priority at a time when this process was the only decisive factor in the prognosis of war casualties. [START_REF] Horne | Triage in the defense medical services[END_REF] The evolution of war medicine has given rise to the need to prioritize access to other scarce resources such as blood products, diagnostic methods and life-saving interventions prior to surgery. As a result, preoperative and pre-hospital categorization tools have been added to classic surgical triage without replacing it.

Most of them were based on physiological data (consciousness, arterial blood pressure, heart and respiratory rates) since they predict both mortality and resource utilization. [START_REF] Eastridge | Admission physiology criteria after injury on the battlefield predict medical resource utilization and patient mortality[END_REF][START_REF] Cancio | Prediction of mortality and of the need for massive transfusion in casualties arriving at combat support hospitals in Iraq[END_REF] Based on the analysis of radial pulse and consciousness, FTS is the most simple illustration of this rationale. 17 NATO preferred using the START algorithm based on four variables: walking ability, breathing, radial pulse, and ability to execute a simple order. [START_REF] Super | START: Simple triage and rapid treatment plan[END_REF] This algorithm has been adopted with minimal adjustments by the British and Australian rescue services and the most recent result of its use in Middle Eastern conflicts is the MPTT. [START_REF] Vassallo | Major incident triage: Derivation and comparative analysis of the Modified Physiological Triage Tool (MPTT)[END_REF] The later was derived from a large cohort of 6,095 war casualties managed consecutively at Camp Bastion in Afghanistan, and prospectively validated on a cohort of 354 war casualties in the same center, demonstrating a lower undertriage rate (16%) than comparable triage tools. [START_REF] Vassallo | Major incident triage: Derivation and comparative analysis of the Modified Physiological Triage Tool (MPTT)[END_REF] However, these war triage tools were built and tested for hospital triage by physician and not prehospital triage. As no triage policy has ever given complete satisfaction to its end-users, it is very likely that they will continue to evolve, mainly to distinguish very quickly the most urgent category of casualties, i.e. avoiding undertriage. This tends to create a simple pre-hospital categorization into only two groups.

The dichotomous scale (AE/RE) corresponds to that taught in France after previous terrorist attacks, [START_REF] Carli | Terrorism in France[END_REF] in line with the more recent recommendations of simplicity. [START_REF] Aylwin | Reduction in critical mortality in urban mass casualty incidents: analysis of triage, surge, and resource use after the London bombings on July 7[END_REF] This simple tool is widely used in France by paramedics and firemen, as well as media and justice officers. [START_REF] Fournier | Attentats de Paris : dans les hôpitaux parisiens, "la plupart des blessés avaient été traversés de balles, c'était horrible[END_REF][START_REF] Molins | Attentats à Paris : l'intégralité des événements, par le procureur de Paris (François Moulins)[END_REF] The reference standard (expert panel) was appropriate when considering that no admission to ICU, blood transfusion, embolization, or death was noted in casualties sorted as RE, and no discharge on day 1 was noted in casualties sorted as AE. Our study shows that using this simple categorization, 78% of the casualties of a multisite terrorist attack were effectively sorted in the prehospital settings, indicating that it was effectively applied despite the very high number of casualties and persistence of the threat. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] This method seems more adapted to these circumstances than those conventionally used for prehospital triage like the FTS, START or MPTT. In the setting of a terrorist attack, these tools proved to be too complicated to use because they require applying an algorithm, measuring vital parameters and filling in a triage tag. Such a complex process is probably unrealistic in the confusion created by a large-scale terrorist attack, in a dangerous environment where emergency teams are under extreme pressure to stop bleeding and evacuate quickly the victims while facing the threat of continuing terrorist attacks. Consequently, during massive shootings, pre-hospital triage can be under-used or not used at all with the risk of important overtriage since the rescue teams will tend, for safety, to transport all the casualties with penetrating trauma only to a level 1 trauma center to the detriment of level 2 or 3 centers which could be less overcrowded and adapted to the care of RE if they had been appropriately categorized. The AE/ER binary triage limiting overtriage seems then adapted to mass casualties with penetrating injuries during terrorist attacks. The proportion of undertriage and overtriage observed during the Paris attacks (36 and 8%, respectively) should be compared to the best one reported by trained surgeons during war conditions (16 and 28%, respectively). [START_REF] Cancio | Prediction of mortality and of the need for massive transfusion in casualties arriving at combat support hospitals in Iraq[END_REF] In the Paris massive event, overtriage was limited, preventing hospital saturation, but undertriage was higher than those observed with other scores. Only 12 AE casualties needed secondary transport, only one of them to a level 1 trauma center. Moreover, the AE/RE method did not adversely interfere with the care of patients since the mortality observed during a massive event was comparable to routine care in traumatology.

It should be pointed that this triage process does not apply to the "hot zone" where tactical medical units intervene. [START_REF] Park | How to stop the dying, as well the killing, in a terrorist attack[END_REF] The qualitative analysis provided some clues for future improvement. Emphasizing the need for staging thoraco-abdominal lesions as AE and conversely staging limb lesions without severe hemorrhage as RE may easily and further improve the diagnostic performance of prehospital triage (Electronic supplement Table S4). The comparison of the triage diagnostic performance on scene and on arrival at the hospital in a subgroup of casualties showed no significant difference in the proportion of casualties appropriately classified, suggesting that it is reproducible (Table 3).

In France, the AE/ER triage concept has been widely used, not only by professional health care providers but also by the media and live TV channels publishing reports of multiple casualty incidents or terrorist attacks. [START_REF] Fournier | Attentats de Paris : dans les hôpitaux parisiens, "la plupart des blessés avaient été traversés de balles, c'était horrible[END_REF][START_REF] Molins | Attentats à Paris : l'intégralité des événements, par le procureur de Paris (François Moulins)[END_REF] Consequently, it was understood by the public and adopted as common reporting language tool for all (medical and non medical) services involved. In the future, in accordance to the Hartford consensus, [START_REF] Jacobs | Hartford Consensus: A call to action for THREAT, a medical disaster preparedness concept[END_REF] outlining the crucial role of the public to "stop the bleeding", the bystander's use of this simple triage concept could also be considered to improve prehospital care organization. A binary categorization is simple to understand, easy to remember and to implement even during stressful events, it does not require complicated measures because it relies on a global and visual assessment of the wounded, it can be taught quickly and performed by first responders, EMTs and also by more specialized teams including physicians enabling everybody to speak the same language. This simple binary approach is flexible and can be used to assess priority for extraction, emergency care and transport of casualties.

Strengths and limitations

The strengths of our study are related to the large number of casualties and use of diagnostic research methodology. [START_REF] Moll | Challenges in the validation of triage systems at emergency departments[END_REF] Several limitations should be noted. Because this study was retrospective, it suffers from possible bias, particularly the existence of missing data related to the non-standardization of information from medical records. The injuries were less severe than those previously reported in mass casualty events, [START_REF] Gutierrez De Ceballos | Casualties treated at the closest hospital in the Madrid, March 11, terrorist bombings[END_REF][START_REF] Gaarder | The twin terrorist attacks in Norway on July 22, 2011[END_REF] since all casualties were included. [START_REF] Raux | Analysis of the medical response to November 2015 Paris terrorist attacks: resource utilization according to the cause of injury[END_REF] We did not include casualties deceased on scene and thus reported in-hospital mortality among patients that arrived alive at hospital which is significantly lower than the overall mortality of the event (24%). Lastly, data concerning FTS, START, and MPTT were obtained using simulation and not in real conditions and the fact that we used a dichotomous categorization as the reference one may have also biased the comparison.

Conclusion

Analysis of the 2015 Paris area terrorist attacks showed that a simple triage categorization had relatively good diagnostic performance, resulting in no detectable adverse effect when considering mortality. Nevertheless, the qualitative analysis of severe under-and overtriage

Figure 1

 1 Figure 1

Figure 2

 2 Figure 2

Acknowledgement: We thank Dr. David Baker DM, FRCA, (Emeritus Consultant Anaesthesiologist, Department of Anaesthesiology and Critical Care, Hôpital Necker-Enfants Malades, Paris) for reviewing the manuscript and Dr. David Hajage (Department of Biostatistics, Sorbonne Université and APHP) for statistical advice.

indicate some room for further improvement. These elements will make it possible to better adapt management plans for massive events.

Author contributions:

AJ, MR and BR conceived and designed the study. AJ, JPT, MR and BR analysed and interpreted the data. AJ, MR, and BR wrote the initial draft. AJ and BR performed the statistical analysis. All authors subsequently critically edited the report, read and approved the final report. MR had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. MR and BR obtained funding and supervised the study.

Declaration of interest:


Sandeep ; Shrivastava

Parijat Sarkar

Pascal Preira

Laurence Salomé

Amitabha Chattopadhyay

Cholesterol-Dependent Dynamics of the Serotonin 1A Receptor Utilizing Single Particle Tracking: Analysis of Diffusion Modes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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[START_REF] Kwik | Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin[END_REF][START_REF] Tsai | Cholesterol deficiency perturbs actin signaling and glutamate homeostasis in hippocampal astrocytes[END_REF][START_REF] Sun | The effect of cellular cholesterol on membrane-cytoskeleton adhesion[END_REF][START_REF] Sarkar | Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism[END_REF] Importantly, both cholesterol and the actin cytoskeleton have been shown to alter the lateral dynamics of membrane proteins such as G protein-coupled receptors (GPCRs) [START_REF] Pucadyil | G-protein-dependent cell surface dynamics of the human serotonin 1A receptor tagged to yellow fluorescent protein[END_REF][START_REF] Jaqaman | Regulation from within: The cytoskeleton in transmembrane signaling[END_REF][START_REF] Ganguly | Actin cytoskeleton-dependent dynamics of the human serotonin 1A receptor correlates with receptor signaling[END_REF] which serve as major drug targets across all clinical areas. [START_REF] Chattopadhyay | GPCRs: Lipid-dependent membrane receptors that act as drug targets[END_REF][START_REF] Sriram | G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?[END_REF][START_REF] Chan | New binding sites, new opportunities for GPCR drug discovery[END_REF] GPCRs are the largest and most diverse class of proteins in higher eukaryotic plasma membranes that regulate a wide array of signaling processes across the membrane. [START_REF] Chattopadhyay | GPCRs: Lipid-dependent membrane receptors that act as drug targets[END_REF][START_REF] Katritch | Structure-function of the G protein-coupled receptor superfamily[END_REF][START_REF] Sakmar | Introduction: G-protein coupled receptors[END_REF] They play an important role in a large number of physiological processes such as neurotransmission, cellular metabolism, cellular differentiation and immune response. As a result of their involvement in crucial biological processes, GPCRs have emerged as major therapeutic targets [START_REF] Sriram | G protein-coupled receptors as targets for approved drugs: how many targets and how many drugs?[END_REF][START_REF] Jacobson | New paradigms in GPCR drug discovery[END_REF] and account for ~40% of currently approved drugs. [START_REF] Chan | New binding sites, new opportunities for GPCR drug discovery[END_REF] The serotonin 1A receptor, an important neurotransmitter receptor which belongs to the GPCR family, is extensively studied among the serotonin receptors and mediates a gamut of neurological, cognitive and behavioral functions. [START_REF] Pucadyil | The serotonin 1A receptor: a representative member of the serotonin receptor family[END_REF][START_REF] Kalipatnapu | Membrane organization and function of the serotonin 1A receptor[END_REF][START_REF] Müller | Serotonin and psychostimulant addiction: focus on 5-HT 1A -receptors[END_REF][START_REF] Glikmann-Johnston | Hippocampal 5-HT 1A receptor and spatial learning and memory[END_REF][START_REF] Sarkar | Biophysics of serotonin and the serotonin 1A receptor: fluorescence and dynamics[END_REF][START_REF] Albert | The 5-HT 1A receptor: Signaling to behavior[END_REF][START_REF] Sarkar | Structure, dynamics and lipid interactions of serotonin receptors: excitements and challenges[END_REF] Since the serotonin 1A receptor plays an essential role in human physiology, it represents a major therapeutic target in developing drugs against neuropsychiatric disorders such as depression, anxiety and even cancer. [START_REF] Lacivita | 5-HT 1A receptor, an old target for new therapeutic agents[END_REF][START_REF] Fiorino | 5-HT 1A receptor: an old target as a new attractive tool in drug discovery from central nervous system to cancer[END_REF] Lateral diffusion of membrane proteins represents a fundamental physical property that regulates the dynamics of protein-protein and lipid-protein interactions, which in turn regulates their function. [START_REF] Edidin | Getting there is only half the fun[END_REF][START_REF] Sarkar | Insights into cellular signaling from membrane dynamics[END_REF] Membrane proteins often undergo confined lateral diffusion, which arises due to compositional heterogeneity in cell membranes and the actin cytoskeleton, that could span different spatiotemporal scales. [START_REF] Clausen | Visualization of plasma membrane compartmentalization by high-speed quantum dot tracking[END_REF] The presence of an intricate network of actin cytoskeleton below the membrane constitutes a major source of confined lateral diffusion in membranes. [START_REF] Kwik | Membrane cholesterol, lateral mobility, and the phosphatidylinositol 4,5-bisphosphate-dependent organization of cell actin[END_REF][START_REF] Kusumi | Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson's fluid-mosaic model[END_REF][START_REF] Kusumi | Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules[END_REF] Several observations using high-resolution microscopy based approaches have recognized the role of the actin cytoskeleton as a key factor that determines the dynamics of membrane proteins. [START_REF] Ganguly | Actin cytoskeleton-dependent dynamics of the human serotonin 1A receptor correlates with receptor signaling[END_REF][START_REF] Fujiwara | Phospholipids undergo hop diffusion in compartmentalized cell membrane[END_REF][START_REF] Ganguly | Cholesterol depletion mimics the effect of cytoskeletal destabilization on membrane dynamics of the serotonin 1A receptor: A zFCS study[END_REF][START_REF] Shrivastava | Role of actin cytoskeleton in dynamics and function of the serotonin 1A receptor[END_REF] Measurement of membrane dynamics is often challenging due to the inherent noise in cellular systems. A variety of microscopybased techniques have been established to measure diffusion in heterogeneous biological membranes. The widely used approaches to probe lateral dynamics in membranes are fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single particle tracking (SPT). [START_REF] Sarkar | Insights into cellular signaling from membrane dynamics[END_REF][START_REF] Day | Tracking microdomain dynamics in cell membranes[END_REF][START_REF] Chen | Methods to measure the lateral diffusion of membrane lipids and proteins[END_REF][START_REF] Jacobson | The lateral organization and mobility of plasma membrane components[END_REF][START_REF] Sarkar | Exploring membrane organization at varying spatiotemporal resolutions utilizing fluorescence-based approaches: implications in membrane biology[END_REF] In recent years, significant progress has been made toward the measurement and interpretation of membrane dynamics at single-molecule level that helps detection and tracking of individual membrane-bound molecules under physiological conditions with a high spatiotemporal resolution. [START_REF] Lagerholm | Detecting microdomains in intact cell membranes[END_REF][START_REF] Kasai | Single-molecule imaging revealed dynamic GPCR dimerization[END_REF][START_REF] Yu | Single-molecule studies in live cells[END_REF][START_REF] Marguet | Lateral diffusion in heterogeneous cell membranes[END_REF] The advantage of singlemolecule measurement is that it allows to circumvent ensemble averaging from multiple molecules, thereby providing information about the heterogeneous behavior (and transient phenomena) within subpopulations of molecules. [START_REF] Destainville | What do diffusion measurements tell us about membrane compartmentalisation? Emergence of the role of interprotein interactions[END_REF] SPT has emerged as a powerful approach to monitor lateral diffusion and organization of membrane lipids and proteins. 55,60,61,66,68,69 Although single particle tracking has been utilized to measure dynamics of membrane proteins, [START_REF] Lagerholm | Detecting microdomains in intact cell membranes[END_REF][START_REF] Wieser | Tracking single molecules in the live cell plasma membranedo's and don't's[END_REF] the application of SPT to GPCRs is relatively limited. [START_REF] Shrivastava | Role of actin cytoskeleton in dynamics and function of the serotonin 1A receptor[END_REF][START_REF] Kasai | Single-molecule imaging revealed dynamic GPCR dimerization[END_REF][START_REF] Daumas | Confined diffusion without fences of a G-protein-coupled receptor as revealed by single particle tracking[END_REF][START_REF] Veya | Single molecule imaging deciphers the relation between mobility and signaling of a prototypical G protein-coupled receptor in living cells[END_REF] In this work, we explored the diffusion characteristics of the human serotonin 1A receptor in live cells under acute and chronic cholesterol depleted conditions utilizing SPT. Analysis of GPCR diffusion in the membrane helps understand GPCR function and provides insight into cellular signaling. [START_REF] Sarkar | Insights into cellular signaling from membrane dynamics[END_REF] By analysis of diffusion modes, we show how acute and chronic cholesterol depletion could influence diffusion behavior of the serotonin 1A receptor, which could have crucial implications in GPCR function. Cell culture and treatment. HEK-293 cells stably expressing the N-terminal myctagged serotonin 1A receptor (HEK-5-HT 1A R) were generated as described previously. [START_REF] Kumar | Exploring endocytosis and intracellular trafficking of the human serotonin 1A receptor[END_REF] The stock solution of lovastatin was prepared as described previously. [START_REF] Shrivastava | Chronic cholesterol depletion using statin impairs the function and dynamics of human serotonin 1A receptors[END_REF] Cells grown for 24 h were incubated with 2.5 M lovastatin in complete DMEM/F-12 medium for 48 h in a humidified atmosphere at 37 °C with 5% CO 2 . Control cells were grown without lovastatin under similar conditions. Acute cholesterol depletion was carried out using M CD as described previously. [START_REF] Pucadyil | Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin 1A receptor in the plasma membrane of living cells[END_REF]

METHODS

Materials

RESULTS AND DISCUSSION

In this work, we utilized HEK-293 cells stably expressing the N-terminal myc-tagged serotonin 1A receptor (HEK-5-HT 1A R, see Figure 1a) to explore the role of cholesterol on receptor dynamics (lateral diffusion). The serotonin 1A receptor expressed in HEK-5-HT 1A R cells is predominantly localized on the plasma membrane as shown in a representative confocal microscopic image at a mid-plane section of a cell (see Figure 1b). Notably, we previously demonstrated that serotonin 1A receptors heterologously expressed in HEK-5-HT 1A R cells could mimic the pharmacological and cellular function of the native receptors in terms of ligand binding, coupling to G-proteins, cAMP signaling and cellular trafficking. [START_REF] Kumar | Exploring endocytosis and intracellular trafficking of the human serotonin 1A receptor[END_REF][START_REF] Kumar | A molecular sensor for cholesterol in the human serotonin 1A receptor[END_REF] In addition, the N-terminal myc-tag allows tracking the trajectory of the serotonin 1A receptor using SPT by fluorescently labeling the receptor in HEK-5-HT 1A R cells with quantum dots (QD) conjugated to streptavidin which are pre-coupled to a biotinylated anti-c-myc antibody.

Signal transduction mediated by membrane-bound receptors has been proposed to involve differential lateral mobility of various interacting partners in the membrane. [START_REF] Cuatrecasas | Membrane receptors[END_REF][START_REF] Kahn | Membrane receptors for hormones and neurotransmitters[END_REF][START_REF] Peters | Lateral mobility of proteins and lipids in the red cell membrane and the activation of adenylate cyclase by -adrenergic receptors[END_REF] This constitutes the basis of the 'mobile receptor' hypothesis which proposes that lateral diffusion determines the interaction between receptor and effector molecules and subsequent signaling at the plasma membrane. [START_REF] Sarkar | Insights into cellular signaling from membrane dynamics[END_REF] Since signaling via GPCRs involves the functional interaction of effector molecules in a heterogeneous membrane environment, the spatiotemporal dynamics of these components in membranes acts as a determining factor of the signaling output by these receptors. [START_REF] Ganguly | Actin cytoskeleton-dependent dynamics of the human serotonin 1A receptor correlates with receptor signaling[END_REF][START_REF] Sarkar | Insights into cellular signaling from membrane dynamics[END_REF] To explore the role of membrane cholesterol in regulating the diffusion of the serotonin 1A receptor, we depleted cholesterol from HEK-5-HT 1A R cells using both chronic and acute cholesterol depletion methods. The main difference between chronic and acute cholesterol depletion methods is the experimental time scale associated with the two processes. [START_REF] Sarkar | Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism[END_REF][START_REF] Sarkar | Differential membrane dipolar orientation induced by acute and chronic cholesterol depletion[END_REF] Acute cholesterol depletion involves physical removal of cholesterol using carriers such as M CD, a water-soluble carbohydrate polymer with a central nonpolar cavity, that efficiently and selectively extracts membrane cholesterol in a relatively short time (~minutes). [START_REF] Zidovetzki | Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies[END_REF][START_REF] Mahammad | Cholesterol depletion using methyl--cyclodextrin[END_REF] On the other hand, chronic cholesterol depletion is carried out over a longer time period (~days) using cholesterol biosynthetic inhibitors that mimic physiological conditions. [START_REF] Endo | A gift from nature: The birth of the statins[END_REF] Cholesterol lowering agents such as statins are extensively used for chronic cholesterol depletion and represent some of the best-selling oral cholesterol-lowering drugs. [START_REF] Endo | A historical perspective on the discovery of statins[END_REF][START_REF] Goldstein | A century of cholesterol and coronaries: From plaques to genes to statins[END_REF] Statins competitively inhibit the rate-limiting enzyme HMG-CoA reductase in the cellular cholesterol biosynthetic pathway. [START_REF] Istvan | Structural mechanism for statin inhibition of HMG-CoA reductase[END_REF] The short-term diffusion coefficient of the serotonin 1A receptor using the SPT method was found to be similar to the diffusion coefficient obtained for other class A GPCRs such as the neurokinin-1 receptor 72 and the -opioid receptor [START_REF] Daumas | Confined diffusion without fences of a G-protein-coupled receptor as revealed by single particle tracking[END_REF] . The values of average D 1-2 for cells treated with lovastatin and M CD were found to be 3.0 0.3 x 10 -2 m 2 s -1 and 2.5 0.2 x 10 -2 m 2 s -1 , respectively. Such a reduction in diffusion coefficient of membrane proteins upon cholesterol depletion has been reported earlier [START_REF] Veya | Single molecule imaging deciphers the relation between mobility and signaling of a prototypical G protein-coupled receptor in living cells[END_REF][START_REF] Shrivastava | Chronic cholesterol depletion using statin impairs the function and dynamics of human serotonin 1A receptors[END_REF][START_REF] Baier | Cholesterol modulation of nicotinic acetylcholine receptor surface mobility[END_REF][START_REF] Vrljic | Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane[END_REF][START_REF] Kenworthy | Dynamics of putative raft-associated proteins at the cell surface[END_REF] and could be due to the further compartmentalization of the plasma membrane, driven by enhanced actin polymerization under these conditions. [START_REF] Sarkar | Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism[END_REF] In addition, the reduction observed in diffusion coefficient upon acute cholesterol depletion could be due to the inherent regional micro-heterogeneity in the plasma membrane. [START_REF] Vrljic | Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane[END_REF][START_REF] Nishimura | Cholesterol depletion induces solid-like regions in the plasma membrane[END_REF] SPT provides specific information about lateral dynamics of single particles in a wellresolved spatiotemporal scale which is crucial for the study of complex biological membranes. A characteristic feature of SPT measurements is that it allows diffusion analysis of individual trajectories and therefore the distribution of these trajectories among different diffusion modes could be analyzed. In addition, SPT allows to distinguish sub-populations of molecules undergoing different modes of diffusion, which is very useful to understand the micro-heterogeneity (domains) in membranes. We identified four diffusion modes by analysis of video trajectories: random, confined, transiently confined and directed diffusion (Figure 4). Interestingly, upon acute cholesterol depletion (using M CD), the fraction of trajectories characterized by random diffusion mode exhibited reduction. In addition, both chronic and acute cholesterol depletion led to an increase in confined diffusion mode relative to control condition (see Figure 4a). Along with the change in various diffusion modes, we observed significant changes in the diffusion coefficient (D 1-2 ) of the serotonin 1A receptor among different diffusion modes. For example, we observed a significant decrease in D 1-2 for random diffusion mode upon statin treatment (see Figure 4b). On the other hand, D 1-2 for confined population remain invariant whereas D 1-2 for transiently confined mode exhibited a significant reduction upon acute and chronic cholesterol depletion (Figure 4b). These observations are supported by previous reports suggesting that cholesterol depletion results in the rearrangement of the actin cytoskeleton. [START_REF] Vrljic | Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane[END_REF][START_REF] Sun | The effect of cellular cholesterol on membrane-cytoskeleton adhesion[END_REF][START_REF] Sarkar | Chronic cholesterol depletion increases F-actin levels and induces cytoskeletal reorganization via a dual mechanism[END_REF] The generation of these compartments (domains) on the cell membrane could be attributed to the polymerization of the actin cytoskeleton which induces dynamic constraints on receptor diffusion and therefore reduces the population of random diffusion, along with a concomitant increase in confined (or transiently confined) diffusion modes.

The effect of the increase in dynamic constraints upon chronic cholesterol depletion gets further manifested in the serotonin 1A receptor diffusion, as evident from the overall reduction in the distribution of the radius of confinement (R) and an overall shift toward

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The Journal of Physical Chemistry lower values of the confinement radius (see Table 1 and Figure 5). The average radius of confinement was ~213 nm for receptors undergoing confined diffusion in control cells (Table 1). The corresponding values for the average radius of confinement upon chronic cholesterol depletion showed a prominent reduction. The average radius of confinement undergoing confined diffusion values for receptors under chronic cholesterol depleted condition was ~176 nm, resulting in ~17% reduction relative to the control condition (Table 1). The average radius of confinement upon acute cholesterol depletion using M CD displayed no significant decrease (for the confined population). Overall, these results suggest that dynamic constraints on receptor diffusion upon chronic cholesterol depletion increase, which could lead to the formation of small-sized confined zones induced by the actin cytoskeleton.

Interestingly, the transiently confined population showed a significant reduction in radius of confinement upon acute cholesterol depletion (Table 1). This reinforces our previous observations, using beam radius dependent FRAP, that acute cholesterol depletion induces dynamic (transient) confinement of the receptor. [START_REF] Pucadyil | Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin 1A receptor in the plasma membrane of living cells[END_REF] To test whether the observed confinement upon cholesterol depletion was due to change in actin polymerization, we carried out quantification of F-actin under acute and chronic cholesterol depleted conditions using a quantitative high-resolution confocal microscopy based approach previously developed by us that allows measurement of F-actin content using an image reconstruction method. [START_REF] Ganguly | Reorganization of the actin cytoskeleton upon G-protein coupled receptor signaling[END_REF] To quantitatively estimate the extent of Factin reorganization, we labeled F-actin with Alexa Fluor 546 conjugated phalloidin (a specific fluorescent probe for F-actin [START_REF] Faulstich | Fluorescent phallotoxins as probes for filamentous actin[END_REF] ). Figure 6(a-c) shows representative confocal micrographs with maximum intensity projections (MIPs) of the actin cytoskeleton under various treatment conditions. The panels in Figure 6(d-f b The concentration of M CD was 5 mM.

  . BSA, CaCl 2 , methyl--cyclodextrin (M CD), DMSO, doxycycline, Dglucose, EDTA, gentamycin sulfate, MgCl 2 , MnCl 2 , NaHCO 3 , penicillin, poly-L-lysine, streptomycin, Triton X-100 and Tris were purchased from Sigma (St. Louis, MO). Qdot 655 streptavidin conjugate, Alexa Fluor 546 phalloidin and Amplex Red cholesterol assay kit were obtained from Molecular Probes/Invitrogen (Eugene, OR). Anti-myc tag antibody Cterminal (Biotin) was obtained from Abcam (Cambridge, UK). Anti-myc antibody Alexa Fluor 488 conjugate was purchased from Millipore (Bedford, MA). Lovastatin was purchased from Calbiochem (San Diego, CA). DMEM/F-12 (Dulbecco's modified Eagle medium: nutrient mixture F-12 (Ham) (1:1)), hygromycin and fetal calf serum were obtained from Invitrogen/Life Technologies (Grand Island, NY). Water was purified through a Millipore (Bedford, MA) Milli-Q system and used throughout.

  Briefly, after growing cells for 3 days in complete DMEM/F-12 medium, cells were incubated in serum-free DMEM/F-12 medium for 3 h in a humidified atmosphere at 37 °C with 5% CO 2 . Cholesterol depletion was performed by treating cells with 5 mM M CD in serum-free DMEM/F-12 medium for 30 min in a humidified atmosphere at 37 °C with 5% CO 2 , followed by washing with PBS. See Supporting Information (Section S1) for more details. Receptor localization using confocal microscopic imaging. HEK-5-HT 1A R cells were seeded at a density of ~10 4 cells on 22x22 mm glass coverslips and grown for 3 days as described earlier. 58 Serotonin 1A receptors were labeled with the anti-myc antibody Alexa Fluor 488 conjugate and imaged using an inverted Zeiss LSM 880 confocal microscope (Jena, Germany) with a 488 nm argon laser and emission was collected from 500 to 560 nm. See Supporting Information (Section S2) for more details. Estimation of cellular cholesterol content. Amplex Red cholesterol assay kit was used to estimate cholesterol content from cell lysates obtained from control, 2.5 M lovastatintreated and 5 mM M CD-treated cells. 76 Cholesterol content values were normalized to total cellular protein estimated using the BCA assay. 77 Single particle tracking experiments. Serotonin 1A receptors in HEK-5-HT 1A R cells were labeled with antibody conjugated quantum dots (QDs) for SPT measurements. A Cascade II 512 EM-CCD camera (Roper Scientific, Tucson, AZ) operating at 25 Hz acquisition frequency on a Zeiss Axio-observer A1 microscope (Jena, Germany) at room temperature (~22 °C) was used to track the trajectories of QDs at the cell surface. The Multiple Target Tracing program developed by Sergé et al. 78 was used to analyze the trajectories of all QDs in a video sequence. The diffusion coefficient inside the confined trajectory segments and the size of the domains (or radius of confinement (R)) were determined by fitting MSD(t) with its theoretical expression for confined diffusion. 79 See Supporting Information (Section S3) for more details. F-actin labeling of cells. F-actin labeling in HEK-5-HT 1A R cells under control (without treatment), 2.5 M lovastatin-treated and 5 mM M CD-treated conditions was performed using Alexa Fluor 546 phalloidin as described previously. 32,80,81 See Supporting Information (Section S4) for more details. Quantitation of F-actin. F-actin was imaged using an inverted Zeiss LSM 510 Meta confocal microscope (Jena, Germany). F-actin quantitation in HEK-5-HT 1A R cells under control (without treatment), 2.5 M lovastatin-treated and 5 mM M CD-treated conditions was performed as described previously. 32,80,81 See Supporting Information (Section S5) for more details.

Figure 2

 2 shows the cholesterol content in cell lysates from cholesterol-depleted HEK-5-HT 1A R cells. The cellular cholesterol content exhibited reduction upon treatment with lovastatin and M CD. For example, cholesterol content was reduced to ~40% of control (without treatment) in lysates from HEK-5-HT 1A R cells treated with 5 mM M CD. HEK-5-HT 1A R cells treated with 2.5 M lovastatin show ~7% reduction in cholesterol content relative to control cell lysates. To ensure the reliability of the single-particle tracking measurements, we optimized the experimental conditions before acquiring the systematic video sequences. We first tested the specificity of QDs for labeling the N-terminally myc-tagged serotonin 1A receptor. We observed fluorescence signal from ~20 QDs per cell when HEK-5-HT 1A R cells were incubated with streptavidin-conjugated QDs pre-coupled to biotinylated anti-c-myc antibody. These QDs remained bound even after washing the cells several times. In contrast, we found only 2 to 3 QDs on each cell in the case of labeling with QDs alone (without prior coupling to biotinylated anti-c-myc antibody). We recorded a series of 80 s video sequences for each cell with varied antibody/QD ratios to calculate the short-term diffusion coefficient (D 1-2 , where "1-2" refers to the first two points of the MSD. In accordance with previous literature, 90 we chose antibody/QD ratio of 1:10 for our SPT experiments to minimally perturb receptor diffusion (more detailed examination of labeling artifacts requires further investigation). We could obtain analyzable tracks from those QDs that were present on top of the cells. Data acquisition was carried out in control (without treatment), 2.5 M lovastatin-and 5 mM M CD-treated HEK-5-HT 1A R cells using these conditions. As a first step, we performed a global analysis of all trajectories collected for each condition. Figure 3 shows the distributions of the short-term diffusion coefficient D 1-2 for control and cholesterol-depleted conditions. As shown in the figure, we observed a shift in the distribution of D 1-2 toward lower values for lovastatin-and M CD-treated cells relative to the control condition. The average D 1-2 value for control cells was 4.4 0.3 x 10 -2 m 2 s -1 (data represent means ± SE).

  ) represent iso-surface maps (contours obtained by joining voxels of equal fluorescence intensity across all z-sections) of cellular F-actin corresponding to the projected images shown in panels (a-c) in Figure 6. To quantitatively estimate cellular F-actin content, we normalized the fluorescence volume enclosed by the iso-surface in each case to the projected area of cells. This ratio (fluorescence volume/area of cells) is a measure of the F-actin content in cells and is shown in Figure 6(g). As shown in the figure, HEK-5-HT 1A R cells treated with lovastatin resulted in ~11% increase in cellular F-actin levels, while M CD treatment show negligible change in F-actin levels. These results are in overall agreement with our recent observations in CHO-K1 cells where we showed that F-actin content significantly increases upon chronic cholesterol depletion but not in acute cholesterol depletion. 32 In this overall backdrop, the effect of the cross-talk between cholesterol and actin cytoskeleton by quantitatively measuring changes in F-actin content as a consequence of acute and chronic cholesterol depletion on the dynamics of the serotonin 1A receptor assumes relevance. CONCLUSION We utilized SPT to measure diffusion behavior of the serotonin 1A receptor under cholesterol-depleted conditions. The advantage of SPT measurements is its ability to distinguish sub-populations of receptors undergoing various modes of diffusion which yields useful information about complex micro-heterogeneous environments such as biological membranes. Our results show that the overall short-term diffusion coefficient of the serotonin 1A receptor exhibits reduction upon both acute and chronic cholesterol depletion (Figure 3). Notably, we observed a significant increase in the confined receptor population under cholesterol-depleted conditions. Such a change in the sub-populations of the serotonin 1A receptor undergoing various modes of diffusion upon cholesterol depletion could have implications on receptor function. These results provide one of the first systematic studies of diffusion behavior of the serotonin 1A receptor using two kinetically different methods of depletion of cholesterol and provides information about the sub-populations of the receptor undergoing various modes of diffusion. Interestingly, our results show that the nature of confinement varies depending on the method of cholesterol depletion. This is due to the fact that whereas chronic cholesterol depletion results in permanent confinement involving remodeling of the actin cytoskeleton, acute cholesterol depletion leads to transient confinement. In addition, this work highlights the role of cholesterol and actin cytoskeleton on the dynamics of the serotonin 1A receptor which could be helpful in understanding the correlation between dynamics and function of the serotonin 1A receptor. We conclude that measurements of receptor dynamics at varying spatiotemporal scales could provide novel insight toward developing a conceptual framework for cellular signaling. SUPPORTING INFORMATION Section S1, Cell culture and treatment; Section S2, Confocal microscopic imaging of receptor localization; Section S3, Single particle tracking experiments; Section S4, F-actin labeling of cells; Section S5, Fluorescence microscopy and F-actin quantitation ACKNOWLEDGMENTS This work was supported by Indo-French Centre for the Promotion of Advanced Research (Project Number 4603-2) to A.C. and L.S. A.C. gratefully acknowledges support from SERB Distinguished Fellowship and CSIR Bhatnagar Fellowship. P.S. was supported as a Senior Project Associate by a CSIR FBR grant (MLP 0146) to A.C. We gratefully acknowledge members of the Chattopadhyay laboratory and Drs. Bhagyashree Rao (Crick Institute) and G. Aditya Kumar (University of Michigan) for their comments and suggestions. Page 14 of 36 ACS Paragon Plus Environment The Journal of Physical Chemistry

Figure 1 .

 1 Figure 1. Features of the human serotonin 1A receptor in HEK-293 cells. (a) A schematic representation of the topological features of the serotonin 1A receptor with a myc-tag (maroon) at its N-terminal. The membrane is shown as a bilayer of phospholipids (headgroup shown in blue) containing cholesterol (shown in red). (b) A representative confocal microscopic midplane section image of the serotonin 1A receptor stably expressed in HEK-293 cells (HEK-5-HT 1A R cells). Serotonin 1A receptors were labeled with anti-myc antibody conjugated to Alexa Fluor 488 (green) and the nucleus was labeled with DAPI (blue). The scale bar represents 10 m.

Figure 2 .

 2 Figure 2. Cholesterol content of lysates from HEK-5-HT 1A R cells. Cholesterol content was estimated in lysates from HEK-5-HT 1A R cells treated with 2.5 M lovastatin or 5 mM M CD (** and *** correspond to significant (p < 0.01 and p < 0.001) difference in cholesterol content in lovastatin-and M CD-treated cells, respectively, relative to control cells). Values are normalized to cholesterol content in control (untreated) cells. Data represent means ± SE of three independent experiments.

Figure 3 .HEK- 5 -

 35 Figure 3. Distribution of diffusion coefficient of serotonin 1A receptors determined by SPT. Distribution of short-term diffusion coefficient (log D 1-2 ) calculated from the trajectories of serotonin 1A receptors obtained by SPT in control (blue circle, n = 77), 2.5 M lovastatintreated (red triangle, n = 101) and 5 mM M CD-treated (inverted green triangle, n = 102) HEK-5-HT 1A R cells. The time duration for the video recordings was set to 80 sec and other details are as described previously.[START_REF] Shrivastava | Role of actin cytoskeleton in dynamics and function of the serotonin 1A receptor[END_REF]

Figure 4 .

 4 Figure 4. (a) Relative distribution of SPT trajectories of serotonin 1A receptors among the four diffusion modes. The relative distribution of SPT trajectories of serotonin 1A receptors in HEK-5-HT 1A R cells among the four diffusion modes observed in control (blue bars, n = 77), 2.5 M lovastatin-treated (red hatched bars, n = 101) and 5 mM M CD-treated (green criss-

Figure 5 .

 5 Figure 5. Distribution of domain radii of confinement zones. Histograms for distribution of domain radii of confinement in the confined trajectories measured by SPT for serotonin 1A receptors in (a) control (blue bars, n = 48), (b) 2.5 M lovastatin-treated (red bars, n = 53) and (c) 5 mM M CD-treated (green bars, n = 62). All other details are as described previously. 58 See Methods for other details.

Figure 6 .

 6 Figure 6. Reorganization of actin cytoskeleton in HEK-5-HT 1A R cells upon cholesterol depletion. The actin cytoskeleton of HEK-293 cells was labeled with Alexa Fluor 546 phalloidin. F-actin content was estimated using a quantitative high-resolution confocal microscopic technique previously developed by us. 81 Panels (a-c) represent maximum intensity projections obtained from confocal z-sections under control, 2.5 M lovastatintreated and 5 mM M CD-treated conditions, respectively. The respective iso-surfaces corresponding to the z-sections are shown in panels (d-f). The F-actin content values in control, lovastatin-and M CD-treated cells are shown in panel (g). Data represent means ± SE of at least 11 independent measurements from three independent experiments (** corresponds to significant (p < 0.01) difference in F-actin content in cells treated with 2.5 M lovastatin relative to control cells). See Methods for other details.

  Figure 1 Shrivastava et al.

  Figure 3 Shrivastava et al.

  Figure 4 Shrivastava et al.

  Figure 6 Shrivastava et al.

Table 1 Diffusion parameters of the serotonin 1A receptor for different diffusion modes __________________________________________________________________________ Diffusion modes _________________________________________________________

 1

		Random	Confined	Directed	Transiently Confined
					______________________
					Random	Confined
	___________________________________________________________________________
	(a) Control
	D 1-2 (10 -2 m 2 /s)	4.6 ± 0.5	3.4 ± 1.1	1.9	5.0 ± 1.6	4.2 ± 0.5
	# of trajectories	21	6	2	48
	R (nm)		213 ± 8			287 ± 30
	(b)

Lovastatin-treated a D 1-2 (10 -2 m 2 /s) 3.2 ± 0.3 2.7 ± 0.7 2.4 ± 0.7 3.2 ± 0.5 2.9 ± 0.4 # of trajectories 29

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			15	4	53
	R (nm)		176 ± 27			263 ± 31
	(c) M CD-treated b
	D 1-2 (10 -2 m 2 /s)	3.5 ± 0.9	2.1 ± 0.3	2.3	2.2 ± 0.4	1.9 ± 0.2
	# of trajectories	11	27	2	62
	R (nm)		203 ± 26			214 ± 22
	_________________________________________________________________________________________
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a The

concentration of lovastatin was 2.5 M.

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