Morgane Flégeau

Hélène Soubelet

Sophie Carré

Sébastien Barot

Xavier Lagurgue

Sabine Bognon

Philippe Clergeau

What evidence exists on the possible effects of urban forms on terrestrial biodiversity in western cities? A systematic map protocol

Keywords: Urban biodiversity, Infra-city scale, Ecological connectivity, Density

Background: As urban areas expand, scientists now agree that the city is an important space for biodiversity conservation. Yet, still relatively little is known about how urban forms could have a differential impact on terrestrial species and ecosystems. If some reviews have been conducted to examine the link between biodiversity and urban characteristics at an infra-city scale, none have explored the relationship between urban organization and biodiversity and tried to assess the capacity of various urban forms to maintain and possibly favour flora and fauna in the city. The resulting map will present the state of knowledge regarding possible relationships between urban forms and its features on the establishment and settlement of terrestrial and temperate biodiversity at infra-city scale in western cities.

Methods:

The systematic map will follow the Collaboration for Environmental Evidence (CEE) Guidelines. We will collect the relevant peer-reviewed and grey literature in French and English language. The scientific literature will be retrieved with the use of a search string in two publication databases, one environmental and one social science database (Web Of Science Core Collection, and Cairn.info). We will also perform supplementary searches (search engines, call for literature, search for relevant reviews). All references will be screened for relevance using a three-stage process, according to a predefined set of eligibility criteria. Our study will concentrate on urban areas at the infra-city scale in cities of the temperate biogeographical zone. The subject population will include terrestrial species and ecosystems, except for archaea and bacteria. The exposure will consider all types of urban forms described by any urban descriptors or measures including heterogeneity, fragmentation, housing density, organisation of urban matrix, urban fabric) and all types of urban features (e.g. size, age of the buildings, materials, urban artefacts). All relevant outcomes will be considered (e.g. species richness, abundance, behaviour). We will provide an open-access database of the studies included in the map. Our results will also be presented narratively, together with tables and graphs summarising the key information coded from the retained articles (e.g. study characteristics, types and areas of research that has been undertaken, types of exposure, population concerned, etc.).

Background

Today, the majority of the world's population is concentrated in cities and the surfaces occupied by such urbanized land continue to expand [START_REF]World population prospects 2019: Highlights[END_REF]. Researchers now agree that urban spaces are important for biodiversity conservation [START_REF] Aronson | Biodiversity in the city: key challenges for urban green space management[END_REF][START_REF] Lepczyk | Biodiversity in the city: fundamental questions for understanding the ecology of urban green spaces for biodiversity conservation[END_REF]. Among other reasons, nature and biodiversity in cities are known to be essential for human health

Open Access

Environmental Evidence *Correspondence: morgane.flegeau@fondationbiodiversite.fr 1 French Foundation for Biodiversity Research (FRB), 195, rue Saint Jacques, 75005 Paris, France Full list of author information is available at the end of the article and well-being [START_REF] Fuller | Psychological benefits of greenspace increase with biodiversity[END_REF][START_REF] Keniger | What are the benefits of interacting with nature?[END_REF]. As a consequence, the greening of cities is promoted by urban policies, especially in their densely populated parts, to make them more liveable. However, researches show that access to green space is frequently associated with higher socio-economic classes, who tend to live in less dense neighbourhoods [START_REF] Boone | Parks and people: an environmental justice inquiry in Baltimore, Maryland[END_REF][START_REF] Ferguson | Contrasting distributions of urban green infrastructure across social and ethno-racial groups[END_REF]. As a result, greening and density policies may be seen by planners as difficult to reconcile [START_REF] Haaland | Challenges and strategies for urban greenspace planning in cities undergoing densification: a review[END_REF]. As cities develop their biodiversity policies, more information concerning the impacts of different urban forms on terrestrial biodiversity is critical for urban planning and management to achieve specific desired biodiversity outcomes.

The characteristics of the urban environment provide specific conditions for establishment of biodiversity. By using the word "biodiversity", we include here all terrestrial species and terrestrial ecosystems except for archaea and bacteria. The extent of impervious surfaces is one of the specificities of urban context. Species richness declines and animal and plant species composition changes as urbanization intensifies [START_REF] Mckinney | Effects of urbanization on species richness: a review of plants and animals[END_REF][START_REF] Concepcion | Impacts of urbanisation on biodiversity: the role of species mobility, degree of specialisation and spatial scale[END_REF]. Research on urban biodiversity often considers cities through an urbanization gradient, from rural to urban-or vice versa, with the measurement of a proportion of impervious surface to assess the impact of urban environment on ecosystems. If it's practical to evaluate and intuitive [START_REF] Beninde | Biodiversity in cities needs space: a meta-analysis of factors determining intra-urban biodiversity variation[END_REF] this type of indicator is often insufficient to explain ecological conditions in cities [START_REF] Alberti | The effects of urban patterns on ecosystem function[END_REF]. A city is also characterized by its spatial complexity and its great diversity from an architectural and historical point of view. At the infracity scale, various urban forms are superimposed on the urbanization gradient. Therefore, studies focusing on the urbanization gradient tend to simplify a much more complex reality.

Examining the forms of the built environment has been mostly the subject of various disciplines such as architecture, urban planning, or geography [START_REF] Kropf | Morphological Investigations: Cutting into the Substance of Urban Form[END_REF][START_REF] Marshall | Streets and patterns[END_REF][START_REF] Moudon | Urban morphology as an emerging interdisciplinary field[END_REF][START_REF] Panerai | Analyse urbaine[END_REF]. Depending on the classifications and criteria used to describe them, urban forms range from the densest inner-city spaces to the least crowded peri-urban transition zone. Urban forms follow several approaches, which correspond to various spatial scales and historical processes [START_REF] Raimbault | Generating urban morphologies at large scales[END_REF]. The multiplicity of the definitions and the variety of indicators used to define them can lead to confusion [START_REF] Ahmadian | Sustainable cities: the relationships between urban built forms and density indicators[END_REF]. Here we chose to consider urban forms at the infra-city scale. Indeed, urban forms encompass physical characteristics in parts of the city and other nonphysical aspects, such as density. Due to resource limitations, we considered temperate western cities and their various urban forms (see Fig. 1 for examples). Studies on cities and urban areas often simplify the ecological processes in their body of work. Therefore, they provide few useful insights to understanding relationships between urban forms and terrestrial biodiversity.

In ecology, urban typologies can be described through composition and spatial configuration. It refers to landscape ecology theories [START_REF] Wu | Landscape ecology[END_REF]. Composition is the ratio of buildings in relation to green spaces and the second is the spatial arrangement of these items among themselves [START_REF] Pellissier | Birds are also sensitive to landscape composition and configuration within the city centre[END_REF]. These configurations are the result of natural characteristics of the site and of historical, architectural, and cultural conditions in which the city was created and expanded. For each urban form, there are specific spatial organisations in terms of parcel grids, roads and builtup islands, urban fabrics, types of buildings, and public spaces [START_REF] Panerai | Formes urbaines: de l'îlot à la barre[END_REF]. It is then important to consider elements like width and length of the roads, size, age and orientation of the buildings, gauges, compactness, alignments or setbacks in relation to the street.

Many studies have focused on the different urban characteristics and their configuration in the urban context. Indeed, the configuration of green spaces in the city-including the organization of urban features, including green spaces (e.g. parks, urban woods, semi natural areas) and ecological corridors-can have a positive influence on habitats, on plants, and on animal species. They could also have a differentiated impact on flora and fauna at infra-city scale [START_REF] Beninde | Biodiversity in cities needs space: a meta-analysis of factors determining intra-urban biodiversity variation[END_REF]. Other ecological studies have focused on certain types of urban forms, as suburban landscapes and showed that their spatial organisation can be important to biodiversity [START_REF] Marzluff | Conserving biodiversity in urbanizing areas: nontraditional views from a bird's perspective[END_REF]. Moreover, some authors have addressed how different types of land use influence animal and plant species [START_REF] Fernandez-Juricic | Avifaunal use of wooded streets in an urban landscape[END_REF][START_REF] Smith | Urban domestic gardens (V): relationships between landcover composition, housing and landscape[END_REF]. In 2018, Jokimaki et al. highlighted that the densest urban areas can even harbour threatened bird species [START_REF] Jokimaki | Urban core areas are important for species conservation: a European-level analysis of breeding bird species[END_REF].

Yet, little seems to be known about the comparison of the capacity of different urban forms to preserve, maintain, or sustain biodiversity and ecosystems and ecosystem services. Some reviews have been carried out on the link between biodiversity and urban features at the infracity scale [START_REF] Mckinney | Effects of urbanization on species richness: a review of plants and animals[END_REF][START_REF] Beninde | Biodiversity in cities needs space: a meta-analysis of factors determining intra-urban biodiversity variation[END_REF][START_REF] Nielsen | Species richness in urban parks and its drivers: a review of empirical evidence[END_REF][START_REF] Urbanova | How to support avian diversity in an urban landscape: a bibliography[END_REF]. In 2000, a review of the scientific literature showed that the differentiated impact of urban forms on fauna and flora was never formally tested in scientific works [START_REF] Alberti | Urban form and ecosystem dynamics : empirical evidence and practical implications[END_REF]. This topic is at the interface between urban ecology and urban studies, and also between the science and practice of urban planning and architecture. We may wonder about the impact of various types of urban morphologies, such as small collectives scattered over a large plot, residential areas with houses centred on their gardens, or even buildings arranged in closed blocks on biodiversity.

Furthermore, if greening the city may provide important ecosystem services to city dwellers, increasing biodiversity in cities therefore brings an element of sustainability to the system. Indeed, respecting the functioning of natural ecosystems (food chains, dispersion processes, mutualism…) may allow for the reduction in management costs of green spaces, and for compensate any climatic or sanitation accident that could drive the disappearance of established species. Promoting biodiversity must therefore imply not only a diversity of species but also relationships between them and their environment. Taking into account ecological corridors or sufficient surfaces for species directly questions the way the urban organization is designed. By exploring the relationship between urban form and terrestrial biodiversity in the scientific literature, we thus aim to assess the capacity of urban forms to maintain animal and plant species in the city and also to provide answers to urban planners of temperate western cities to conduct urban projects including biodiversity.

Stakeholder engagement

This work is part of a broader program named BAUM (Biodiversity, Land use planning and Morphology), launched in 2019 by an interdepartmental agency of the French government, the PUCA (Plan Urbanism Construction Architecture). This program explores the possibility of reconciling the densification of buildings with the preservation of biodiversity in European cities. The results of this systematic map conducted on the links between terrestrial biodiversity and urban forms in temperate western cities will be able to enlighten the stakeholders on issues such as the impact of urban sprawl in France. BAUM program is supervised by a Scientific Council, composed of researchers, civil servants in charge of national public policies and various stakeholders from the private sector.

The Scientific Committee was consulted to suggest relevant literature (scientific and grey literature), specialist website and also eligibility criteria for article screening.

Within the Scientific Committee, a review team has been constituted. It is composed of the two project managers and three experts (SC, PC, SB, HS and MF): Finally, this review team is completed by two other researchers specializing in ecology (SB) and in architecture (XL).

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Objective of the review

The objective of this systematic map is to gather and synthesize studies that explore whether different types of urban development (exposure) can have a differential impact (outcomes) on terrestrial species and ecosystems (population).

Primary question

What is the range and extent of evidence on the effects of urban forms and features on the establishment of terrestrial and temperate biodiversity at the infra-city scale, in urban areas of Europe, North America, New Zealand, and Australia?

Methods

This review strictly follows the CEE guidelines [START_REF]Guidelines and standards for evidence synthesis in environmental management[END_REF] and conforms to the ROSES reporting standards [START_REF] Haddaway | ROSES flow diagram for systematic reviews[END_REF] [see Additional file 1].

Searching for articles Languages

Due to limited resources and also to the languages understood by the review team, only studies published in English and in French will be included in this systematic map. A large body of scientific literature is available in English and research reports and grey literature are accessible in French.

Search string

The search string based on PECO (Population, Exposure, Comparator, Outcomes) keywords in English was built based on a scoping exercise conducted in "Web of Science Core Collection" database in June 2019. A detailed description of the PECO elements that we used for the systematic map is presented in Table 1.

Search terms were initially identified from articles of a test-list and then discussed and validated by the review team. They were organized into four blocks [see Additional file 2 search string complete with excluded words].

• Block A: descriptors of ur context • Block B: descriptors population • Block C: descriptors outcomes • Block D: descriptors exposure

In each block, search terms were combined using the Boolean operator "OR" and the four blocks were combined using the Boolean operator "AND" i.e., ' A' AND 'B' AND 'C' AND 'D' . This search string was structured in four elements related to Context, Population, Outcomes and Exposure:

A (Context) AND B (Population) AND C (Outcomes) AND D (Exposure).

It was adopted to limit the number of results, because of time and human resources limitations, after some preliminary searches. The search string was built by successive iterations, starting with the whole list of key words and refined to maximise the comprehensiveness of the equation with the test list. OR geometr* OR gradient* OR habitat OR "habitat diversity" OR "habitat patch*" OR heterogene* OR "housing density" OR "intermediate disturbance" OR island* OR landscape* OR land-cover* OR land-use* OR LULC OR matrix OR microhabitat OR mosaic OR NDVI OR parcel* OR "patch area" OR "patch size" OR patch* OR pattern* OR plot* OR "polycentric development" OR "public green space" OR "renaturation" OR sector* OR shape OR simplif* OR "spatial diversity" OR "spatial heterogeneity" OR "spatial pattern*" OR "stepping-stone" OR structure OR "transit-oriented development" OR "urbanization level" OR "urban fabric" OR "urban sprawl" OR "urban renewal" OR wasteland OR composition OR configuration OR organization)).

Comprehensiveness of the search

To check the performance of the search strategy, a test list of 30 scientific articles (presented in Additional file 3) was initially obtained from experts of the review team, from various existing reviews, by searching on Google, and in grey literature sources.

On the basis of the criteria listed above, we chose to do the bibliographic search on two databases:

• "Web of Science Core Collection" on the Web of Science platform (Clarivate) to which the review team had access via the team's members institution (INRA subscription of HS). As explained above, a scoping exercise was conducted using this database.

Our search string was run on 6th of June 2019 and covered SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, ESCI and CCR-EXPANDED, without any timespan restriction. • "Cairn.info" to which the review team had access via the team's members institution. This social sciences database includes studies published in French and in English. Another scoping exercise has been conducted. The search string was made using the following terms: (urban form OR urban morphology OR infra-city OR city OR town) AND (biodiversity OR animals OR plants). Our search string was run on 7th of June 2019 without any timespan restriction.

A total of 29,523 articles were listed in both databases. 26,672 of them were referenced in WOS CC and 2851 of the articles of the test list were indexed in Cairn. Consequently, combining the two databases, the search comprehensiveness was 100% [see Additional file 3].

Bibliographic databases

We first listed the peer-reviewed databases to which the members of our review team had access, databases that covered ecology and also humanities and that guaranteed reproducibility. The following publication databases have been listed and will be included in the search strategy:

• WOSCC • Cairn.info (https:// www. cairn. info/) • Persée (https:// www. persee. fr/) • Archinform(https:// www. archi nform. net/) • Urbamet (http:// www. urbam et. com/) • ArchiRès (https:// www. archi res. archi. fr/) Due to the project's resource limitations (in terms of time, and financial resources), we were not able to search more than two web-based search engines. Because of the position of the subject at the science-society interface, we chose to explore at least one environmental database and one social science database. This choice is consistent with the number of articles obtained during the scoping exercise.

Web-based search engines

Additional searches will be performed using Google Scholar search engine. The search will be limited to the first 200 results according to the limited effectiveness of this web-based academic search engine for evidence reviews [START_REF] Haddaway | The role of google scholar in evidence reviews and its applicability to grey literature searching[END_REF]. The relevant peer-reviewed articles in English will be collect, without any timespan restriction, using the search terms combined:

Urban form AND biodiversity.

Supplementary searches

To maximize the coverage of results, different sources of literature will be investigated since the topic of this systematic map straddles the science and practice of urban planning. The aim of collecting grey literature is therefore to provide the broadest possible overview of professional and operational productions in the urban planning field. The search will be carried with the help of an intern during one month in order to collect writings of the stakeholders of public institutions, design offices, associations. The search for grey, professional, and operational literature will be carried out through Internet searches. A broader search will be made using the following French terms:

(« Forme urbaine» OR « Morphologie urbaine») AND biodiversité.

Indeed, this broader search for grey literature will be conducted in French (language spoken by the review team) since the research program including the systematic map is a French ministry agency. We identified a preliminary set list of sources [see Additional file 4].

Specialist websites

We will search for additional literature in French with the following search terms: (Forme urbaine OR Morphologie urbaine) AND biodiversité.

The following websites of French specialist organizations will be searched for relevant publications: The resources will be collected by browsing the document collections of public institutions, academic institutions and associations, and consulted on the Google search engine.

A call for grey literature will be conducted through the Scientific Council of our project to find non-peerreviewed literature, including reports published in French, articles from professional journals of urban planning and architecture. All members of the Scientific Council have been asked to provide documents that they considered relevant to the subject.

Duplicate removal

All articles will be exported into different collections using the reference management software Zotero ® and merged into one database after the export. All duplicates will be removed manually in Excel. We will not use review management software to screen and code literature.

Article screening and study eligibility criteria

Screening process

All the articles obtained by the search process will be screened according to a three-stage selection procedure. The screening of titles, abstracts, and full-texts will be performed by two reviewers independently (MF and HS). The four other members of the review team will help during the abstract screening stage (SC, SB, XL, SB and PC). If there is disagreement between the two primary reviewers (MF and HS), the inclusion or exclusion of the article will be discussed with all members of the review team.

A Kappa test will be performed by all the reviewers (HS, MF, SC, SB, XL, SB, and PC) at each screening stage. A random sample of 10% of the titles (after duplicate removal), 10% of the retained abstracts, and 10% of the retained full-texts will be selected and each of the seven reviewers will screen this sample independently. The operation will be repeated until reaching a Kappa score ≥ 0.6. Any disagreements will be identified and discussed between the reviewers. The criteria for rejection of an article will be decided, discussed, and validated by the review team.

During the scoping stage conducted in the "Web of Science Core Collection", the three stages of the screening process were tested by one reviewer (MF) in order to refine the eligibility criteria. A second reviewer will examine the rejected articles during each sorting stage to assess the consistency of the inclusion/exclusion decisions. Thus, an a posteriori cross check will determine if articles are re-included or not.

The details of the final decision of inclusion or exclusion of articles selected at the full-text screening stage (with the reason of exclusion) will be provided in an Excel database [see Additional file 5].

Eligibility criteria

Eligibility criteria were developed in consultation with the review team at each stage of the screening process. In any case of doubt regarding the presence of a relevant inclusion criterion or if there is insufficient information to make an informed decision, articles will be retained for assessment at a later stage. The articles accepted based on their titles but without any abstracts will pass by default to the full-text screening stage.

To be included in our systematic map, studies must attain the criteria of exposure detailed in Table 2. We used increasingly precise criteria from the first screening step to the last in order to retain articles that could have otherwise been excluded because of a lack of precision/ detail in the title or in an abstract, concerning the study scale.

Title

Inclusion criteria

Firstly, all titles will be retained if presence both of any terms related to cities or urban environment and terrestrial species (wild animal and plants) or ecosystems.

Exclusion criteria

If there is absence of explicit mention of urban or biodiversity related terms in title, the articles will be excluded.

Abstract

Inclusion criteria

Articles will be retained if terms related to infra-city district scale, urban features are present, and if the study design includes a spatial comparison (alternative urban forms) or a temporal comparison (previous urban forms).

Exclusion criteria

Articles will be excluded if urban context or urban forms are treated in a vague manner or even unspecified. An abstract will also be rejected if the urban category studied relates to articles treating urban functions (e.g.: commercial, industrial or residential for example). This also includes management of biodiversity and functions of infrastructure and architectural elements. For instance, if an article deals with the impact of lamp posts on bird communities, the screening will be done on whether it is part of an urban composition (and will be considered as part of the "form" category) or whether it is considered through production of light and therefore as a disturbance for animal species.

Full text

Inclusion criteria

During screening of full-texts, a distinction will be made based on a higher degree of accuracy of the description of the urban exposure. We will focus on articles addressing elements of urban infrastructure (e.g. material, architecture) or elements (e.g. streets, avenues) and, in particular different types of urban forms (e.g. collective district or low-density area). The final screen will be based on urban descriptors and features, preferably quantitative. Articles that accurately describe the urban descriptors in their results will be selected and will be the most relevant articles for our subject. These articles will include at least two quantified urban descriptors.

To truly focus on urban forms at the infra-city scale, we will apply a final exclusion criterion, discussed and validated by the review team. We will exclude the articles addressing the interface between spaces supporting biodiversity linked to their urban environment (connectivity, fragmentation, permeability of the matrix, etc.) and the urban matrix. This category includes articles dealing with various types of green spaces and their interface with urban context.

Study validity assessment

No study validity assessment will be performed because the intention of the map is not to examine the robustness of the study designs. However, our data coding strategy will include information regarding the design of each study, The data produced could be used further by users of this systematic map to assess the evidence presented in the article.

Data coding strategy

Key data from the selected articles after the three screening stages will be extracted based on the PECO structure using an a priori specified Codebook, a predefined spreadsheet that was discussed and validated by the review team [the articles used to assess the effectiveness of the data coding strategy are listed in Additional file 6]. Each article will be coded based on the full text using keywords and expanded comments fields describing various aspects of the study [See the codebook in Additional file 5]. In case studies lack detail on the information required or if details need to be clarified during the review process, the corresponding authors will be contacted by email.

Study description

• Basic bibliographic information (authors, title, journal, publication date, etc.) • Study site, country of the study and region Each article selected at full-text stage will be double coded by two members of the review team to ensure the consistency and accuracy of data extraction. If this type of coding is not possible due to limited resources, four members of the review team will independently carry out an a posteriori crosscheck on the meta-data of 10 publications randomly chosen at the first stage of the screening process. To test agreement between reviewers, Kappa scores will be calculated. Any disagreement will be discussed and the eligibility criteria will be revised to improve the data coding strategy. This process will be carried out until reaching Kappa score ≥ 0.6.

Table 3 Categories of exposure to code in the systematic map

Categories of exposure Description Example reference from test list or scoping exercise

Morphological characteristics Description of street widths and its spatial organization, building heights [START_REF] Murgui | Factors influencing the bird community of urban wooded streets along an annual cycle[END_REF] Connectivity Connectivity between urban items and vegetated areas (green spaces, urban gardens, remnants, etc.) [START_REF] Bierwagen | Predicting ecological connectivity in urbanizing landscapes[END_REF] Urban development scenarios Use of urban or ecological modelization [START_REF] Tannier | Assessing the capacity of different urban forms to preserve the connectivity of ecological habitats[END_REF] Urban projects Comparing spaces before and after the construction of urban projects [START_REF] Hostetler | Post-construction effects of an urban development on migrating, resident, and wintering birds[END_REF] Urban density Study of sectors characterized by a variable density of housing, buildings and population [START_REF] Soga | Land sharing vs land sparing: does the compact city reconcile urban development and biodiversity conservation? Rhodes J, editor[END_REF] Major types of urban areas Either large sectors such as "dense urban", "suburban", etc., in terms of degree of compactness or land-use patterns or description of the urban form through function (e.g. residential, business areas, industrial) [START_REF] Andersson | Understanding how built urban form influences biodiversity[END_REF] Urbanization gradient Description of types of urban form even if the discriminating element is the percentage of urban land use by impervious surfaces [START_REF] Avondet | Drosophila (Diptera : Drosophilidae) response to changes in ecological parameters across an urban gradient[END_REF] Study mapping and presentation

We will produce an open-access database (Microsoft Excel sheet) including all the studies that meet our inclusion criteria and coded metadata. It will be included as an appendix to the systematic map. Where there is more than one study included in an article, each study will be recorded as a specific entry in the database i.e. one line per study. There will be several lines for each article if necessary.

The systematic map report will include summary figures and tables. Possible knowledge gaps (un-or underrepresented subtopics that warrant further primary research) and knowledge clusters (well-represented subtopics for full synthesis by a systematic review) will be identified e.g. by cross-tabulating key meta-data variables in heat maps (e.g. biological groups and outcomes). We will use our categorization of urban descriptors and outcomes to identify where there is a lack of knowledge and for which species. Interactive maps will be produced to present the data according to urban forms and make them more accessible and easily viewed. We will also pay attention to study designs and to the comparators used in the body of literature collected. Based on these results, recommendations will be made on priorities for future research on the effect of urban form on biodiversity.

Fig. 1

 1 Fig. 1 Illustration of various urban forms in western cities

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  Secondary questions: • What are the categories of urban forms most studied? What are the variables most used to describe urban features? • What are the most commonly described outcomes? • In which countries have the most studies been conducted? • What research fields study this interface topic? • What are the most used study designs?

Table 1 PECO elements PECO Description Population

 1 All terrestrial species and terrestrial ecosystems except for archaea and bacteria in cities of the temperate biogeographical zone (according to Olson & Dinerstein (1998) classification of biomes: temperate broadleaf and mixed forests, temperate coniferous forests and Mediterranean forests, woodlands and scrub). Due to resource limitations, we only considered temperate western citiesExposureAll types of urban forms described by any urban descriptor or measure including heterogeneity, fragmentation, housing density, organisation of the urban matrix, urban fabric) and all types of urban features (e.g. size, age of the buildings, materials, urban artefacts). A first list of urban forms and features expected to be found in the literature has been discussed within the scientific committee of BAUM. To provide a frame to this work, the keywords have been categorized into four groups. They are related to urban forms, urban morphology and density, process of urbanization, functions and finally to street furniture. They are described below: -Urban form: allotment, architecture, boulevard, centre, collective, dwelling, district, historical centre, intra-urban, metropolis, neighbourhood, peri-urban, public green space, road, suburb, urban fabric, wasteland -Urban morphology: arrangement, block, complexity, connectivity, continuous, disturbance, heterogeneity, dimension, discontinuous, division, fragmentation, heterogeneity, mosaic, shape, structure Density: compactness, gradient, urbanization level, land cover, land use, housing density -Urbanization process: urban sprawl, urban renewal, pattern, polycentric development, renaturation -Urban functions: commercial, residential, industrial Street furniture: bench, lamppost and pavement All types of urban forms will be considered and described Comparator Alternative urban forms or previous urban forms Outcomes Any descriptors of presence or absence of the population including, but not restricted to, abundance of species, diversity of species, and behaviour of species. Outcomes studied will include: ecosystem services, functional characteristics, functional composition, functional feature, species richness, abundance, amount, assemblage, colonization, dispersion, diversity ecological process, function, habitat, index, management of soil, migration number, population, presence, proportion, population structure, viability Context All urban areas at the infra-city scale (scale of observation of the urban form)

Table 2

 2 Inclusion and exclusion criteria for the title screening

		Inclusion criteria	Exclusion criteria
	Population	All terrestrial species (wild animal and plants) and ecosystems	Freshwater, marine, and coastal biodiversity and ecosystems
			Micro-organisms (archaea and bacteria)
	Exposure	All types of urban features (e.g. materials used, size or age of	Other types of exposure in the urban environment (e.g. physico-
		buildings) or urban form (e.g. historical centre, residential area).	chemical and climatic exposure)
		A first list of exposure is identified here:
		-Urban form: allotment, architecture, boulevard, centre, collec-
		tive, dwelling, district, historical centre, intra-urban, metropolis,
		neighbourhood, peri-urban, public green space, road, suburb,
		urban fabric, wasteland
		-Urban morphology: arrangement, block, complexity, con-
		nectivity, continuous, disturbance, heterogeneity, dimension,
		discontinuous, division, fragmentation, heterogeneity, mosaic,
		shape, structure
		Density: compactness, density, gradient, urbanization level,
		land cover, land use, housing density
		-Urbanization process: urban sprawl, urban renewal, pattern,
		polycentric development, renaturation
		-Urban functions: commercial, residential, industrial
		Street furniture: bench, lamppost and pavement
	Outcomes	All outcomes related to the included populations including, but	Measures of genetic, physico-chemical or chemical variables (e.g.:
		not restricted to, biology/physiology (e.g. body size, growth),	climate, temperature, carbone storage)
		space use (e.g. species distribution, individual movements),
		ecosystem composition (e.g. species richness, abundance) and
		species behaviour (e.g. reproduction, predation)
	Context	Intra-urban or city scale in cities of the temperate biogeograph-	-Reference to rural context;
		ical zone according to Olson & Dinerstein (1998) classification	-National or regional scale;
		of biomes: temperate broadleaf and mixed forests, temperate	-All other biogeographical zones (e.g.: tropical, boreal)
		coniferous forests and Mediterranean forests, woodlands and
		scrub
	Language	Any article written in English or in French
	Type of document Peer-reviewed journal article, book chapter, reports, PhD thesis,	Non-peer-reviewed articles, editorial material, letters or news
		conference abstract and presentation, grey literature	item, posters
	Type of content	In situ studies and modelling studies	Reviews and meta-analyses

Acknowledgements

The authors thank Romain Sordello and Joseph Langridge for providing useful comments on the protocol, Barbara Livoreil for her advice at the onset of this work, regarding the methodology of systematic reviews and Camille Huberts for her help with the search for grey literature. We also want to thank the anonymous reviewers as well as the editor for their precious comments and suggestions during the peer-review process.

Funding

This map was funded by the French Ministry of Ecological and Solidarity Transition and the French Fundation for Research on Biodiversity.

Availability of data and materials

All data generated or analysed during this study are included in this article and its Additional files.

Supplementary Information

The online version contains supplementary material available at https:// doi. org/ 10. 1186/ s13750-021-00243-x.

Additional file 1. ROSES systematic map protocols checklist.

Additional file 2. Search string complete. Details of the scoping exercise performed to build the search string.

Additional file 3. Test-list. List of 30 articles selected to assess the effectiveness of the search string.

Additional file 4. Set list of the grey literature sources identified.

Additional file 5.

Codebook of the study. Presentation of the criteria used to data that will be extracted in the systematic map.

Additional file 6. List of 29 articles selected to assess the effectiveness of the data coding strategy.

Authors' contributions

This systematic review protocol is based on a draft written by MF and all authors contributed to the final version. All authors read and approved the final manuscritp.

Declarations

Ethics approval and consent to participate Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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È Eric

Mabrouk Kamel

Dupuis Alain

Smida-Rezgui Sophia

Altafaj Xavier

Grunwald Didier

Platel Jean-Claude

Andreotti Nicolas

Marty Isabelle

Sabatier Jean-Marc

Ronjat Michel

De Waard Michel

Pathologies

Michel De Waard
email: <michel.dewaard@ujf-grenoble.fr>

Transduction of the scorpion toxin maurocalcine into cells. Evidence that the toxin crosses the plasma membrane Est ve

Keywords: Amino Acid Sequence, Biological Transport, Biotinylation, Calcium, chemistry, Carrier Proteins, chemistry, Cell Differentiation, Cell Line, Cell Membrane, drug effects, metabolism, Endocytosis, Endoplasmic Reticulum, metabolism, Gene Products, tat, metabolism, Humans, Kinetics, Microscopy, Confocal, Models, Molecular, Molecular Sequence Data, Muscle, Skeletal, cytology, metabolism, Peptides, chemistry, Protein Conformation, Protein Isoforms, Protein Transport, Ryanodine, metabolism, Ryanodine Receptor Calcium Release Channel, chemistry, Sarcoplasmic Reticulum, metabolism, Scorpion Venoms, metabolism, pharmacokinetics, Signal Transduction, Temperature, Time Factors

Maurocalcine (MCa) is a 33 amino acid residue peptide toxin isolated from the scorpion palmatus. External Scorpio maurus application of MCa to cultured myotubes is known to produce Ca release from intracellular stores. MCa binds directly to the 2+ skeletal muscle isoform of the ryanodine receptor, an intracellular channel target of the endoplasmic reticulum, and induces long-lasting channel openings in a mode of smaller conductance. Here, we investigated the way MCa proceeds to cross biological membranes in order to reach its target. A biotinylated derivative of MCa was produced (MCa ) and complexed with a fluorescent b indicator (streptavidine-cyanine 3) in order to follow the cell penetration of the toxin. The toxin complex efficiently penetrated in various cell types without requiring metabolic energy (low temperature) or implicating an endocytosis mechanism. MCa appeared to share the same features as the so-called Cell-Penetrating Peptides (CPP). Our results provide evidence that MCa has the ability to act as a molecular carrier and to cross cell membranes in a rapid manner (1 2 min) making this toxin the first demonstrated example of a scorpion toxin that translocates into cells.

Maurocalcine (MCa) is a 33-mer toxin isolated from the venom of the scorpion palmatus. Since its initial isolation ( ), Scorpio maurus 1

MCa was successfully produced by chemical synthesis which allowed the characterization of its biological activity and the solution of its 3-D structure ( ). MCa was shown to be one of the most potent effectors of the skeletal muscle ryanodine receptor type 1 (RyR1), an 2 intracellular calcium channel target. The toxin stimulates the binding of H -ryanodine onto RyR1 present in sarcoplasmic reticulum (SR)

[ 3 ]
vesicles ( , ). It induces strong modifications in the gating of RyR1 channels reconstituted in lipid bilayers that are characterized by the 3 4 appearance of long-lasting sub-conductance states ( , ). MCa application to purified SR vesicles produces Ca release which is consistent 4 5 2+ with these biophysical effects ( , ). Using a calcium imaging approach, it was also shown that the addition of MCa to the extracellular 3 4 medium of cultured myotubes induces Ca release from the SR into the cytoplasm ( ). Taken together, these data are coherent with a 2+ 4 direct effect of MCa on RyR1. These observations were confirmed with the identification of MCa s binding site on RyR1 which indicates a ' cytoplasmic localization ( ). 6

According to the H -NMR analysis of MCa, the peptide presents three disulfide bridges (Cys -Cys , Cys -Cys , and Cys -Cys )

[ 1 ]
3 17 10 21 16 32 and folds along an Inhibitor Cystine Knot (ICK) motif ( ). MCa contains three -strands: from amino acid residues 9-11 (strand 1), 20-23 2 β (strand 2) and 30-33 (strand 3), respectively. The -strands 2 and 3 form an antiparallel -sheet. It is a highly basic peptide that contains

β β
eleven basic amino acid residues (33 of the molecule composition). Interestingly enough, eight of the eleven basic residues are oriented % towards the same face of the molecule ( ). Fig. 1 A Two pieces of evidence suggest that MCa should be able to cross the plasma membrane to reach its intracellular target. First, MCa has biological activity consistent with the direct activation of RyR1 when added to the extracellular medium. Second, a structural analysis of MCa reveals a stretch of positively charged amino acid residues that is reminiscent of the protein transduction domains (PTD) found in proteins like Tat from the HIV-1 virus ( , ), the homeotic transcription factor ANTP (encoded by the gene) ( ), 7 8 Drosophila antennapedia 9

and the herpes simplex virus type 1 (HSV-1) VP22 transcription vector ( ). Synthetic peptides corresponding to these domains are called 10 cell-penetrating peptides (CPP) and have in common to contain many basic residues (arginine and lysine), often oriented towards the same face of the molecule ( ). Because of this structural feature, CPP possess the interesting ability to cross biological membranes in a Fig. 1 B receptor-or transporter-independent manner ( ). These peptides seem to target the lipid bilayer directly using complementary charges 11-13 and penetrate the cell through a mechanism called translocation which remains unclear. Three possible models have been put forth in the literature to explain peptide penetration: 1) the inverted-mycelle model ( ) with its membrane-destabilization model variant ( ), 2) the 14 carpet model ( ), and the pore-formation model ( ). Regardless of the precise mode of entry, CPP possess unique features worth 15 16 mentioning: i) delivery in 100 of the cells ( ), ii) delivery in all cell types both and ( , ), iii) ability to cross the % 17 in vitro in vivo 18 19 blood-brain barrier ( ), and iv) the possibility to act as carriers and to translocate large compounds, such as proteins and oligonucleotides 18 ( ). 20

The current study was meant to investigate the ability of MCa to penetrate into cells. We show that a biotinylated version of MCa effectively and rapidly enters various cell types and can behave as a good carrier to allow the penetration of larger compounds. As for other CPP, the mechanism of cell penetration is not linked to an endocytotic pathway. We conclude that MCa represents a new class of disulfide-linked CPP and is the first scorpion toxin shown to enter cells.

Materials and Methods

Chemical Synthesis of MCa and biotinylated MCa

Fmoc-L-amino acids, 4-hydroxymethylphenyloxy resin, and reagents used for peptide synthesis were obtained from Perkin Elmer N-α Life Sciences.

Fmoc-L-Lys(Biotin)-OH was purchased from Neosytem Groupe SNPE. The MCa and biotinylated MCa (MCa ) were N-α b obtained by the solid-phase peptide synthesis ( ) using an automated peptide synthesizer (Model 433A, Applied Biosystems Inc.). 21

Peptide chains were assembled stepwise on 0. ionization time-of-flight mass spectrometry.

Formation of the MCa -streptavidin-cyanine 3 complex b

Soluble streptavidin-cyanine 3 (Strept-Cy3, Amersham) was mixed with four molar equivalent of MCa (1 mM) for 2 hrs at room b temperature in phosphate buffered saline (PBS).

Preparation of heavy SR vesicles

Heavy SR vesicles were prepared following a modified method of Kim and collaborators ( ) as described previously ( ). Protein 22 23 concentration was measured by the Biuret method.

Cell cultures

L6 cells

Rat myogenic L6 cells were obtained from the European Collection of Animal Cell Cultures ECACC (clone C5) and cultured in Dulbecco s modified Eagle s medium supplemented with 15 foetal bovine serum (Life technologies, Inc.) and 1 penicillin/streptomycin ' ' % % (Invitrogen). Differentiation of L6 cells was induced by a shift to differentiation medium (DMEM 5 Horse serum) when they reached + % confluence, as described previously ( ). 24

CA1 hippocampal neurons

Hippocampuses from neonatal mice (between days 1 and 2 post partum) were dissected, cleaned of meninges and placed in Hanks balanced salt solution (HBSS; Invitrogen). They were transferred into a dissociation medium containing HBSS, 1 penicillin/streptomycin % 3 11

(Gibco), 2000 units/ml DNase I and 1 (w/v) trypsin/EDTA and incubated at 37 C for 7 min. After sedimentation, the supernatant was % °removed

and the tissue was washed with a HBSS medium containing 1 penicillin/streptomycin. The tissue was gently triturated in HBBS % medium containing 2000 units/ml DNase I, 10 foetal bovine serum and 1 penicillin/streptomycin with a plastic pipette until a % % homogeneous suspension was obtained. After centrifugation at 100 g during 1 min, the cell pellet was resuspended in Neurobasal/B27 medium (Gibco) containing 0.5 mM L-glutamine and 1 penicillin/streptomycin. Cell cultures were seeded at a density of 10 cells/cm

% 5 2
on culture dishes coated previously with 20 g/ml poly-L-lysine at 37 C for 2 hrs. After 2 days, 3 M cytosine arabinoside was added to μ °μ the cultures to control the proliferation of non-neuronal cells, and 24 hrs later half of the medium was replaced. The culture media were then subsequently changed every other day.

HEK293 cells

Human embryonic kidney cells (HEK293, Gibco) were passaged by 1 trypsin treatment prior to confluence and maintained in % DMEM containing 10 (v/v) foetal bovine serum and 1 penicillin/streptomycin (Invitrogen) and were placed into an incubator and 5

% % %

CO . The culture medium was changed every other day.

[ 3 H -ryanodine binding assay

] Heavy SR vesicles (1 mg/ml) were incubated at 37 C for 2.5 h in an assay buffer composed of 5 nM H -ryanodine, 150 mM NaCl, 2

°[3 ]
mM EGTA, 2 mM Ca ( Ca 5), and 20 mM HEPES, pH 7.4. 100 nM MCa or MCa /streptavidine-Cy3 were added prior to the addition 2+ p = b of heavy SR vesicles. H -ryanodine bound to heavy SR vesicles was measured by filtration through Whatmann GF/B glass filters

[ 3 ]
followed by three washes with 5 ml of ice-cold washing buffer composed of 150 mM NaCl, 20 mM HEPES, pH 7.4. H -ryanodine

[ 3 ]
retained on the filters was measured by liquid scintillation. Non-specific binding was measured in the presence of 20 M unlabeled μ ryanodine. The data are presented as the mean S.E.M. Each experiment was performed in triplicate and repeated at least twice.

±

Ca Release Measurements

2+

Ca release from heavy SR vesicles was measured using the Ca -sensitive dye, antipyrylazo III. The absorbance was monitored at

RESULTS

Synthesis of biotinylated MCa and complex formation with Cy3-labeled streptavidine

We have previously identified MCa as a potent activator of the skeletal muscle RyR1 calcium channel ( , ). As RyR1 is an 3 4 intracellular target, these observations suggested that MCa may modulate RyR1 by one of two mechanisms: (i) indirectly through binding onto a plasma membrane receptor (implies no cell penetration) or (ii) directly through binding onto RyR1 itself (implies cell penetration).

With the recent identification of a RyR1 binding site for MCa ( ) and earlier evidence that MCa alters the gating of purified RyR1 6 channels reconstituted in lipid bilayers, we favour the hypothesis that MCa can reach its intracellular target directly by crossing the plasma membrane. To investigate this question, we synthesized a biotinylated version of MCa (MCa ) in order to create a fluorescent complex b MCa coupled to Cy3-labeled streptavidine (MCa /Strept-Cy3). We first tested whether biotinylation and complex formation with b b

Strept-Cy3 does not impair the functional effects of MCa on RyR1. demonstrates that the MCa /Strept-Cy3 complex retains the Fig. 2 b ability to stimulate H -ryanodine binding on SR vesicles (panel A) as well as to induce Ca release from SR vesicles (panel B). The The data demonstrate that all cells are labelled, with a strong and uniform staining at the plasma membrane and the cytoplasm, whereas the nucleus is weakly labelled ( ). In contrast, cells incubated in the presence of 100 nM non-biotinylated MCa Fig. 3 and equivalent concentrations of Strept-Cy3 do not display any labelling demonstrating that Strept-Cy3 alone is not able to transduce into cells. The association of Strept-Cy3 with MCa is thus required for the fluorescent complex to enter cells, demonstrating the active b function of MCa in this process. These data thus indicate that MCa not only acts as a CPP, but, like many other CPP, has the ability to cargo large molecular weight proteins (streptavidine is 14.6-fold larger than MCa).

Kinetics of cell penetration by the MCa /Strept-Cy3 complex b

To further characterize the transduction properties of MCa, we analyzed the time course of the fluorescence labelling of living non-differentiated L6 cells by MCa /Strept-Cy3 complex. In order to achieve this study, we used time-lapse confocal microscopy in a way b that preserves cell integrity and viability over at least a 1 hr period. Cell labelling is evident for periods of time as short as 3 min after the addition of 100 nM MCa /Strept-Cy3 complex in the extracellular medium ( ). We defined three regions of interest (ROI) ). At the Fig. 4 C start of application of 100 nM of the MCa /Strept-Cy3 complex in the bath, the fluorescence intensity increases in all cell compartments b albeit with different rates. The evolution is fastest and greatest for the plasma membrane (ROI-1) and reaches a peak in 10 min. A slower, but nevertheless important, increase in fluorescence is also observed for the cytoplasmic compartment (ROI-2), while a much smaller, but above background, fluorescence increase is observed for the nucleus (ROI-3). The rate and the relative intensity at which fluorescence increases in each compartment is consistent with the direction of progression of the MCa /Strept-Cy3 complex within the cell, i.e. from the b extracellular space to the plasma membrane, from the plasma membrane to the cytoplasm, and then from the cytoplasm to the nucleus. Of note, the fluorescence signal recorded in ROI-1 corresponds to the fluorescence associated to the plasma membrane plus the cytoplasmic area close to the membrane. Since the signal is normalized, it underestimates the plasma membrane associated fluorescence. In contrast, the relative fluorescence intensity of the cytoplasm should be more accurate. Two conclusions should be drawn from these data. First, the passage of the MCa /Strept-Cy3 complex from the cytoplasm to the nucleus is very weak suggesting that this transition is far less favoured b than the two other ones. Second, the fluorescence associated to cell compartments is far greater than the fluorescence from the bath demonstrating that cells act as MCa concentrators . We also followed the evolution of the fluorescence once the MCa /Strept-Cy3 Several cell mechanisms are known that may allow the entry of MCa. Transduction mechanisms are postulated to be energy-independent and do not require a specific receptor for translocation across the plasma membrane, whereas endocytosis and pinocytosis are energy-dependent. Although a contribution of an endocytotic process is possible, two reasons suggest that it is not the primary mode of entry of MCa. First, the distribution of the MCa /Strept-Cy3 complex in the cytoplasm is quite uniform. Second, the b normal intracellular target of MCa (RyR1) requires a direct release of MCa into the cytoplasm which is hardly compatible with endocytosis. We nevertheless investigated the contribution of energy-dependent processes in the entry of the MCa /Strept-Cy3 complex ( b ). We first tested the effect of decreasing the temperature on the entry of MCa /Strept-Cy3 complex ( ). The data Fig. 5 b Fig. 5 B unambiguously demonstrate that the entry of the complex still occurs at 4 C. At this temperature, the complex similarly labels the plasma °membrane and the cytoplasm. Also, we tested the effect of specific inhibitors of pinocytosis and endocytosis. Neither 3 mM amiloride ( ) nor 50 M nystatin ( ) had any effect on the cell entry of the complex or its relative distribution in the plasma membrane Fig. 5

DISCUSSION

In this manuscript, we demonstrate that MCa has the ability to translocate into cells by crossing the plasma membrane. This process is rapid since translocation appears evident even for incubation times as short as 1 or 2 min. It reaches saturation in the plasma membrane around 10 min, though longer times were required for entry into the cytoplasm. This is consistent with the kinetics of entry of other CPP with a peak of entry of 15 min for the Tat-p27Kip1 ( ) and Tat--galactosidase complexes ( ). 26

β 18
In our experiments, we coupled MCa to streptavidine which is of significantly higher molecular weight than MCa itself, b demonstrating that MCa can also carry large molecules into cells, like other CPP ( , ). The fact that MCa is so efficient with such a 27 28 b large cargo suggests that uncoupled MCa should be even more efficient when added to the extracellular medium of cells. Indeed, it was observed that Ca release from myotubes alone occurred as fast as 3 sec after the addition of MCa, suggesting a much faster translocation 2+ in the absence of a cargo molecule. The demonstration that uncoupled MCa has an even faster kinetics of cell entry awaits the synthesis of a fluorescent derivative of MCa. Attempts to produce such a derivative have been unsuccessful so far. In our experiments, we used a concentration of MCa (100 nM) that is in the range for which other CPP demonstrate efficient translocation (usually between 25 to 200 b nM ( ). We show that translocation of MCa occurs at 4 C, as well as in the presence of inhibitors of pinocytosis and endocytosis, 17

°demonstrating that the process is energy-independent like for other CPP ( , , ). Though the current mechanism of MCa translocation 17 29 30 is unknown, we can only propose that it may occur along one the mechanisms suggested for other CPP. Like for other CPP, we found that maurocalcine has the ability to effectively translocate in different cell types suggesting that the process is insensitive to the nature of the biological membrane or to the types of receptors expressed on its surface. It is thought that CPP interact with the plasma membrane before translocation through their positively charged surface presumably with negative charges present at the extracellular surface of the lipid bilayer ( ). Since MCa also possesses such a positively charged surface, one may assume that it interacts with the lipid bilayer in a 31 manner akin to other CPP. Nevertheless, it should be mentioned that endocytosis may represent an additional contributing factor to MCa cell entry for longer periods of incubation though we did not investigate this question. Referencing the literature, we noticed that all CPP are small peptides. In Tat, the PTD encompasses the basic amino acid residues 47-57 ( , , ), whereas in VP22 and ANTP, the PTD 17 32 33 goes from residues 267-300 ( ) and 43-58 ( ), respectively. Similarly, the putative PTD sequence of maurocalcine should be shorter 29 14 than 33 amino acid residues. The sequence of MCa that triggers translocation is unknown. However, owing to its similarities with other CPP, the basic amino acid rich sequence is the principal suspect. One intriguing observation is that part of the same basic sequence involved in translocation also appears to mediate the interaction with RyR1. For instance, mutation of Arg of MCa, that belongs to the 24 putative CPP sequence, results in a complete loss of interaction with RyR1 ( ). Interestingly, part of the PTD of HIV-1 Tat, the 4

Gly-Arg-Lys-Lys-Arg-Arg sequence, is also a potential nuclear localization sequence ( ). In addition, Tat, VP22 and ANTP are all 12 proteins implicated in transcriptional regulation, and the PTD are the domains that make contact with nucleic acids. This seems to indicate that PTD may intrinsically possess multiple functions: protein translocation (all CPP), protein targeting (Tat), and protein (MCa) or nucleic acid interaction (Tat, VP22 and ANTP).

Overall, we thus have several arguments to believe that MCa behaves as a CPP. Like recognized CPP sequences: 1-MCa is a small peptide, 2-it is positively charged, 3-it enters many cell types, 4-it enters in an efficient manner, 5-at low concentration, 6-the translocation is a fast process, 7-that is energy-independent, and 8-it can carry a cargo molecule. It possesses yet two other essential properties maybe not shared with other CPP which is i) its apparent capacity to enter cells against its concentration gradient and ii) to enter far more rapidly than to exit the cell. The reasons for these properties are unknown but we tentatively assume that the membrane potential may work as a driving force for cell penetration and maintenance. Because MCa is structured by disulfide bridges, it should be classified as belonging to a new class of CPP. Also, the disulfide linkage of MCa, which makes it more rigid than other CPP, implies that the transduction mechanism at the basis of MCa cell penetration does not rely on extensive peptide unfolding. Owing to the growing importance of CPP in delivering functionally important molecules into cells, the identification of MCa will be useful to further understand the molecular basis of cell penetration of CPP. In addition, MCa may constitute a leading compound for the design of new and efficient analogues to deliver active drugs inside cells that would otherwise have limited or no bioavailability. A new CPP may be of interest for biotechnological and pharmaceutical companies. A

The abbreviations used are

  a diode array spectrophotometer (MOS-200 Optical System, Biologic, Claix, France). Heavy SR vesicles (50 g) were actively μ loaded with Ca at 37 C in 2 ml of a buffer containing 100 mM KCl, 7.5 mM sodium pyrophosphate, 20 mM MOPS, pH 7.0, 2+ °supplemented with 250 M antipyrylazo III, 1 mM ATP/MgCl , 5 mM phosphocreatine, and 12 g/ml creatine phosphokinase ( ). Ca

2

 2 by sequential additions of 50 and 20 M of CaCl . In these loading conditions, no calcium-induced calcium release μ 2 interferes with the observations. At the end of each experiment, Ca remaining in the vesicles was determined by the addition of 4 M Ca 2+ μ ionophore A23187 (Sigma) and the absorbance signal was calibrated by two consecutive additions of 20 M CaCl . 2+ μ Imaging of MCa /Strept-Cy3 translocation by confocal microscopy bImaging on fixed cellsCell cultures were incubated with 100 nM (final concentration) of the MCa /Strept-Cy3 complex in the cell culture medium in the dark b and at room temperature unless otherwise stated for 30 min or 1 hr. After three washes with PBS, cells were fixed at room temperature and in the dark by 3 paraformaldehyde during 20 min, washed with PBS and mounted with antifading (Dako) for observation with the % confocal microscope. All conditions were compared under identical settings. Samples were analyzed by confocal laser scanning microscopy using a Leica TCS-SP2 operating system. Cy3 fluorescence was excited by using the 543 nm line of a helium-neon laser, and the fluorescence emission was collected from 554 to 625 nm.Imaging on living cellsLiving cells were incubated at room temperature in fresh culture medium on the stage of an upright compound microscope (Eclipse 600 FN, Nikon) equipped with a water immersion 40 objective (numerical aperture 0.8) and a confocal head (PCM 2000, Nikon) or with × the Leica TCS-SP2 microscop with a 100 objective under XYZt mode. MCa /Strept-Cy3 penetration kinetics were followed × " " b immediately after injection of 100 nM of this complex into the culture medium. Cy3 fluorescence was excited with the 488 nm wavelength line of an argon laser. Emitted light was filtered by a 595 35 nm filter. Images were acquired and analysed with the EZ2000 software ± (Nikon). Relative fluorescence quantitative analysis was realized on the stack using the devoted Leica software.

[ 3 ]Fig. 3 culture

 33 Fig. 3 culture of hippocampal CA1 neurons, and two cell lines, HEK293 and L6 cells (before and after differentiation), were incubated for 30 min (room temperature) with 100 nM of MCa complexed to Strept-Cy3. Cells were then fixed and the fluorescence observed by confocal b microscopy. The data demonstrate that all cells are labelled, with a strong and uniform staining at the plasma membrane and the

Fig. 4 A

 4 Fig. 4 A corresponding to the plasma membrane (ROI-1), the cytoplasm (ROI-2) and the nucleus (ROI-3). The positioning of the different ROI was facilitated by the analysis of the transmitted light image of the cell under observation ( ). We then analyzed the evolution of the Fig. 4 B fluorescence intensity as a function of time in the different ROI with a Leica confocal microscope under the XYZt mode (). At the Fig.4 C

Fig. 4 C

 4 Fig.4C fluorescence intensity of ROI-1 decreases whereas that of ROI-2 increases. ROI-3 fluorescence intensity remains constant. The fluorescence increase of the cytoplasmic compartment must thus be related to a continued transfer of the complex from the plasma membrane into the cytoplasm. This plasma membrane/cytoplasm transfer is probably the rate-limiting step in MCa entry. The decrease in fluorescence intensity of the plasma membrane must thus result both from a leakage of the fluorescent complex to the extracellular medium and from its transfer to the cytoplasm. Longer periods of observations were precluded by the appearance of an inhomogeneous distribution of the fluorescent dye in the cytoplasm, maybe linked to degradation pathways (data not shown). However, we conclude from these data that complexes of MCa /Strept-Cy3 that have penetrated into cells appear to stably remain inside the cell suggesting that MCa b has a favoured direction of plasma membrane crossing.Endocytosis is not required for translocation of the MCa /Strept-Cy3 complex b

C μFig. 5 D

 5 Fig. 5 D and in the cytoplasm, again confirming that the main pathway for entry of MCa into cells does not involve either endocytosis or pinocytosis.

  Fig. 1 3-D solution structure of several peptides. 3-D solution structure of MCa (Protein Data Bank accession code, 1C6W). Basic amino acid residues present on the same face of MCa are shown (Lys , Lys , Lys , Lys , Lys , Arg , Arg and Lys ). Lys belongs to the -strand

β 1 , 30 β

 130 Lys , Lys and Arg to the -strand 2, whereas Lys belongs to the -strand 3. The 3-D solution structures of penetratin 1 code 1KZ0), TAT proteins from HIV1 (1TIV) and equine infectious anemia virus (1TVT) are also shown. Lateral chains of basic amino acid residues are highlighted in blue. The primary structures of the peptides are also provided. The pictures are generated with SwissPDB viewer (Swiss-Prot, Switzerland).

Fig. 2 3 ] 2 addition of 20 M

 23220 Fig. 2 The MCa /Strept-Cy3 complex is functionally active on RyR1. , Stimulation of H -ryanodine binding by 100 nM MCa or MCa b

μ 2 μ. 3

 23 Fig. 3 MCa /Strept-Cy3 complex transduces efficiently in many different cell cultures. 3, 2 and 1 day-old cultures of hippocampal CA1 neurons, b HEK293 cells and non-differentiated L6 cells, respectively, were incubated for 30 min with 100 nM MCa complexed to Strept-Cy3 before b fixation and confocal analysis. Differentiated L6 cells were treated similarly (4-days of differentiation). Fourfold smaller panels represent control experiments in which cells were treated with a mixture of 100 nM non-biotinylated MCa and Strept-Cy3.

Fig. 4

 4 Fig. 4 Kinetic of MCa /Strept-Cy3 complex transduction in non differentiated L6 cells. , Confocal Cy3 fluorescence image of proliferating L6 cell b

Fig. 5

 5 Fig. 5 Cell entry of the MCa /Strept-Cy3 complex is energy-independent. , Control condition for the entry of MCa /Strept-Cy3 complex into b

  25 meq of hydroxymethylphenyloxy resin (1 cross-linked; 0.89 meq of amino group/g)

		%
	using 1 mmol of --Fmoc amino acid derivatives. The side-chain protecting groups were as follows: trityl for Cys and Asn; N α	butyl for tert-
	Ser, Thr, Glu, and Asp; pentamethylchroman for Arg; and N % butyloxycarbonyl or Biotin for Lys. --Amino groups were deprotected by tert-N
	coupled (20 min) as their hydroxybenzotriazole active esters in methylpyrrolidone (4-fold excess). To obtain MCa, aliquot of peptide N-
	resin was removed at its corresponding cycle. After peptide chain assembly, the peptide resin (approximately 0.9 g) was treated between 2
	and 3 hrs at room temperature in constant shaking with a mixture of trifluoroacetic acid/H O/thioanisol/ethanedithiol (88: 5/5/2, v/v) in the 2
	presence of crystalline phenol (2.25 g). The peptide mixture was then filtered, and the filtrate was precipitated by adding cold t
	-butylmethyl ether. The crude peptide was pelleted by centrifugation (3.000 for 10 min), and the supernatant was discarded. The reduced g
	peptide was then dissolved in 200 mM Tris-HCl buffer, pH 8.3, at a final concentration of 2.5 mM and stirred under air to allow
	oxidation/folding (between 50 and 72 h, room temperature). The target products, MCa and MCa , were purified to homogeneity, first by b
	reversed-phase high pressure liquid chromatography (Perkin Elmer Life Sciences, C18 Aquapore ODS, 20 m, 250 10 mm) by means of μ ×
	a 60-min linear gradient of 0.08 (v/v) trifluoroacetic acid/0 30 acetonitrile in 0.1 (v/v) trifluoroacetic acid/H O at a flow rate of 6 % -% % 2
	ml/min ( 230 nm). A second step of purification of MCa and MCa was achieved by ion-exchange chromatography on a carboxymethyl λ = b
	cellulose matrix using 10 mM (buffer A) and 500 mM (buffer B) sodium phosphate buffers, pH 9.0 (60-min linear gradient from 0 to 60%
	buffer B at a flow rate of 1 ml/min). The homogeneity and identity of MCa or MCa were assessed by the following: (i) analytical C18 b
	reversed-phase high pressure liquid chromatography (Merck, C18 Li-Chrospher, 5 m, 4 200 mm) using a 60-min linear gradient of 0.08 μ ×
	%	(v/v) trifluoroacetic acid/0 60 acetonitrile in 0.1 (v/v) trifluoroacetic acid/H O at a flow rate of 1 ml/min; (ii) amino acid analysis -% % 2
	after acidolysis (6 N HCl/2 (w/v) phenol, 20 h, 118 C, N atmosphere); and (iii) mass determination by matrix-assisted laser desorption % °2

α treatment with 18 and 20 (v/v) piperidine/ -methylpyrrolidone for 3 and 8 min, respectively. The Fmoc-amino acid derivatives were

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