Mechanical analysis of infant carrying in hominoids In all higher nonhuman primates , species survival depends upon safe carrying of infants clinging to body hair of adults . In this work , measurements of mechanical properties of ape hair -LRB- gibbon , orangutan , and gorilla -RRB- are presented , focusing on constraints for safe infant carrying . Results of hair tensile properties are shown to be species-dependent . Analysis of the mechanics of the mounting position , typical of heavier infant carrying among African apes , shows that both clinging and friction are necessary to carry heavy infants . As a consequence , a required relationship between infant weight , hair -- hair friction coefficient , and body angle exists . The hair -- hair friction coefficient is measured using natural ape skin samples , and dependence on load and humidity is analyzed . Numerical evaluation of the equilibrium constraint is in agreement with the knuckle-walking quadruped position of African apes . Bipedality is clearly incompatible with the usual clinging and mounting pattern of infant carrying , requiring a revision of models of hominization in relation to the divergence between apes and hominins . These results suggest that safe carrying of heavy infants justify the emergence of biped form of locomotion . Ways to test this possibility are foreseen here . Introduction All higher primates -LRB- except humans -RRB- carry their young clinging to their fur from birth -LRB- Jolly 1972 -RRB- . The correlation between infant carrying and the form of locomotion of adult primates is clear , but no detailed study has focused on the mechanics of the problem . Among nonhuman primates , there is a change in the carrying pattern of infants by adults -LRB- mostly by the mother -RRB- as the infant grows -LRB- Jolly 1972 ; de Vore 1965 ; Goodall 1967 ; MacKinnon 1974 ; Fossey 1979 , 1983 ; Tuttle and Watts 1985 -RRB- . Newborns are carried clinging in close ventro-ventral contact , often with additional support from the mother -LRB- Hoff et al. 1983 -RRB- . Change to infant support over the adult body -LRB- dorsal or lumbar clinging -RRB- occurs some months later for all nonhuman higher primates and extends for years in apes . It seems clear that safety in infant carrying imposes limits on the weight of infants . This work deals with the problem of infant carrying in Hominoids , characterized by increasing body size . Accepted hominoid phylogeny places the branch to the lesser ape gibbon as the oldest . It is followed by the great apes , with the older branch to the arboreal orangutan and the branch originating terrestrial gorilla and chimpanzee coming later , followed by biped hominids -LRB- Yunis and Prakash 1982 ; Ruvolo et al. 1994 ; Lockwood et al. 2004 -RRB- . Mechanical analyses of constraints for safe infant carrying in the usual primate pattern are reported . Measurements of tensile and friction properties of hominoid hairs are presented and conditions for mechanical equilibrium determined . The usual pattern of primate carrying of heavy infants is shown to be incompatible with bipedalism . This brings a new perspective to the hominization process and to the basic changes at the divergence between apes and hominins . Materials and methods Samples For the experimental study of the mechanical properties of ape hairs , three pieces of skin with hairs were obtained from frozen limbs of dead animals at the Laboratoire d'Anatomie Comparée , Muséum National d'Histoire Naturelle , Paris , France -LRB- one gibbon , one orangutan , and one gorilla -- chimpanzee was not available -RRB- . Due to difficulty in obtaining animal skins , the samples came from three individual animals , one piece of about 200 cm2 from each . The three pieces of skin were transported at room temperature immersed in formaldehyde and were treated afterwards by a taxidermist . After the skin was fixed , it became leather , and chemicals were washed out from the hairs , which looked natural . The effect of formaldehyde on hair has been tested in human head hair ; results are presented in the Appendix , and evidence is that no significant change occurs in the relevant tensile parameters . The skin and hair from the different animals studied here are easily recognized by appearance and texture and are species-characteristic . Gibbon hairs are smooth and silky , while orangutan hairs are harsh , hard and very long ; gorilla hair has an intermediate appearance . Estimated values of skin thickness and hair length are given in Table 1 , with other parameters obtained . Table 1Parameter values for apes GorillaGibbonOrangutanSkin thickness -LRB- mm -RRB- ~ 1.5 ~ 0.6 ~ 2.3 Hair Length -LRB- cm -RRB- ~ 6 ~ 4 ~ 10 ∅ -LRB- μm -RRB- 66 ± 2 -LRB- 21 % -RRB- 52 ± 2 -LRB- 19 % -RRB- 120 ± 4 -LRB- 24 % -RRB- α -LRB- N -RRB- 17 ± 2 -LRB- 25 % -RRB- 6.0 ± 0.6 -LRB- 33 % -RRB- 32 ± 1 -LRB- 13 % -RRB- FE -LRB- N -RRB- 0.38 ± 0.03 -LRB- 30 % -RRB- 0.18 ± 0.02 -LRB- 36 % -RRB- 0.49 ± 0.02 -LRB- 12 % -RRB- Fr -LRB- N -RRB- 0.67 ± 0.09 -LRB- 39 % -RRB- 0.29 ± 0.04 -LRB- 47 % -RRB- 0.79 ± 0.04 -LRB- 17 % -RRB- Sr0 .26 ± 0.05 -LRB- 56 % -RRB- 0.28 ± 0.05 -LRB- 52 % -RRB- 0.07 ± 0.01 -LRB- 51 % -RRB- E -LRB- GPa -RRB- 5.0 ± 0.6 -LRB- 42 % -RRB- 2.8 ± 0.3 -LRB- 43 % -RRB- 2.8 ± 0.2 -LRB- 36 % -RRB- Skin thickness and hair length -LRB- estimated -RRB- , hair diameter ∅ , and results obtained from the n = 10 tensile curves for each ape hair . The regression coefficient α and the elastic limit FE are measured in the initial linear Hookean part of the curve , while the force Fr and strain Sr at rupture are measured at the end of the curve . Mean values are given with their standard errors . The Young modulus E is given by α divided by the hair cross-sectional area -LRB- with error propagation -RRB- . The variation coefficient -LRB- CV = 100 × standard deviation/average value -RRB- is given in parenthesis . The three skins provided several samples of different areas with the skin side fixed on pieces of wood , leaving the hairs exposed . The use of taxidermized specimens is the only possibility for simulating the natural situation for friction coefficient measurements . The basic data by Schultz -LRB- 1931 -RRB- on hair density of primates were also obtained from embalmed animals . Methods Several sectioned ape hair cross-sections were examined under a compound microscope -LRB- magnification up to 360 -RRB- , and external appearance as well as hair diameters were measured with a Zeiss Axiovert 200 microscope -LRB- several positions along the hair length -RRB- . Ten single hairs of each of the three animals were cut and analyzed on an automated Instron Tensile Tester , at the standard 65 ± 2 % relative humidity of the air -LRB- RH -RRB- , 20 ± 2 °C and speed 2.6 mm/min . The hair was fixed on one end to a fixed grip and on the other extremity to a movable grip , connected to a pulling force . The defined distance between grips -LRB- Lo -RRB- is between 20 and 50 mm . Curves of force -LRB- given in Newton , MKS unit -RRB- versus longitudinal elongation L -LRB- mm -RRB- were obtained ; the strain , or relative elongation , is given by -LRB- L − Lo -RRB- / Lo . Single human head hair -LRB- with and without immersion in formaldehyde -RRB- was also analyzed as a standard , as a comparison was possible with previous data for human hairs -LRB- Robbins 1994 ; Nikiforidis et al. 1993 ; Franbourg et al. 2003 -RRB- . Twenty single hairs from each of the three animals , still on the skins , were also analyzed and pulled until detachment from the skin occurred . For friction measurements , the wooden pieces with the larger areas from each animal were fixed to a movable inclined plane . The other pieces -LRB- with areas in the interval 16 -- 76 cm2 -RRB- were used in measurements of the static friction coefficient μ -LRB- hairs against hairs -RRB- using variable weights attached to the wood face . The angle of the inclined plane was carefully changed until slipping started at its critical value , and the process was repeated several times to check reproducibility . The RH and the room temperature -LRB- 20 -- 26 °C -RRB- were measured and controlled with a dehumidifier and an air-conditioning system . Results Tensile measurements Mechanical studies of animal fibers have been extensively performed over the last decades because of interest in textile production -LRB- mainly wool , see Feughelman 1997 -RRB- as well as cosmetics -LRB- human head hair , see Robbins 1994 -RRB- . Animal hair is composed of three parts : an external thin cuticle -LRB- protective covering with a scale structure from root to tip -RRB- , a thicker cortex with fibrous proteins , and a central porous medulla , which may be absent in finer hair . Ape hair viewed under an optical microscope are similar in structure to wool and human head hair , as in Fig. 1 , where the external appearance with the cuticle scale structure is shown in Fig. 1a and the cross-section inner structure in Fig. 1b . Fig. 1Micrographs showing orangutan hair , whose light color allows better visualization ; a external appearance with typical cuticle scale structure , b transversal cross section with external cuticle , thicker cortex , and inner medulla Mechanical properties of natural fibers are defined by the cortex of the fiber and are due to the molecular structure of keratin , which constitutes the cortex , as determined many years ago -LRB- Astbury and Street 1931 ; Astbury and Woods 1933 -RRB- . Extensive work during decades evidenced the dependence of mechanical properties essentially on the cross-sectional area of the fiber , thus enabling basic research to be conducted on relatively few single fibers , eliminating the need for statistical methods on a large number of samples -LRB- Feughelman 1997 -RRB- . Tensile measurements give curves of force against strain -LRB- relative elongation -RRB- for a single hair fiber . Force has an important meaning for this study ; stress is obtained dividing the force by the cross-sectional area of the fiber . A typical result with standard human head hair is shown in Fig. 2a and is similar to earlier data on human hair -LRB- Robbins 1994 ; Nikiforidis et al. 1993 ; Franbourg et al. 2003 -RRB- . The curve displays an initial elastic -LRB- Hookean -RRB- linear region , followed by a plastic yield region and a postyield region before breakage . Transition from the elastic to the plastic region corresponds to a first-order transition of the alpha-keratin molecule from the coiled helices to the stretched beta-keratin form -LRB- Bendit 1957 -RRB- , and the plastic region is strongly influenced by the water content -LRB- Feughelman 1997 -RRB- . Fig. 2Measured force × strain -LRB- relative elongation -RRB- curves for hominoid a human , b gorilla , c gibbon , and d orangutan hairs . In a are stressed the initial Hookean linear elastic region -LRB- E -RRB- , the plastic plateau -LRB- P -RRB- , the post-yield region -LRB- PY -RRB- , and the breaking threshold -LRB- B -RRB- . The different curves in b , c , and d correspond to ten hairs of each individual ape . The inset in a is a magnification of the elastic linear region , showing the slope α and the force at the elastic limit FE The linear region extends up to 1 % strain , a result well established for helical alpha-keratins of all animal hairs : the cortex composite has mechanical properties of a single material -LRB- Feughelman 1997 -RRB- . The value of the force at the Hookean elastic limit -LRB- called FE in this paper -RRB- corresponds to the limiting force for full recovery of the fiber after being relaxed . The regression coefficient of the linear region -LRB- called α in this paper -RRB- of the force versus strain curve divided by the cross-sectional area of the fiber gives the Young modulus of elasticity -LRB- E -RRB- of the fiber -LRB- dimension of pressure unit , GPa = 109 N/m2 -RRB- . The values α and FE are determined expanding the initial elastic part of the curve -LRB- which does not , in general , start precisely at zero -RRB- , as seen in the insert in Fig. 2a . Further analysis of human head hair is given in the Appendix . Apparently , no tensile studies on ape hair have been reported . Curves obtained with n = 10 ape hairs of each of the three apes -LRB- one gorilla , one gibbon , and one orangutan -RRB- are shown in Fig. 2b -- d . The curves display all the classical regions of animal hair . Human hair has a larger postyield region and a larger strain at breakage , related to the usual human hair treatment -LRB- frequent washing and combing -RRB- . Maximum stretching is very different in the wet -LRB- 50 % strain -RRB- and dry -LRB- 10 % strain -RRB- states and may be influenced also by hair conditioners -LRB- Robbins 1994 ; Colombera and Joekes 2004 -RRB- . The initial linear region is well defined in all hair curves , and its variability among different hairs of the same ape is due to differences in hair cross-sectional area . The physical parameters α and FE of each of the ape curves were obtained with the procedure shown in the insert of Fig. 2a . Values of force -LRB- Fr -RRB- and strain -LRB- Sr -RRB- at rupture were measured directly at the end of the curves . Table 1 gives statistical averages of parameters obtained from tensile curves -LRB- α , FE , Fr , Sr -RRB- and measured hair diameter ∅ for each species , together with calculated Young modulus of elasticity E , with respective errors . The average values for FE are 60 % -LRB- ± 2 % -RRB- of Fr for the hairs of the three ape species . Hair diameters ∅ in Table 1 are averages over several positions measured along ten single hairs of each ape -LRB- ∼ 50 measurements for each ape -RRB- . One-way analysis of variance -LRB- ANOVA -RRB- test for these ∅ measurements evidenced significant difference between apes -LRB- F2 ,155 = 177 ; p < 0.0001 -RRB- . All tensile parameter values for gorilla hairs , with the only exception being the Young modulus E , are intermediate between gibbon and orangutan hairs . Table 2 gives statistical tests for these four parameters : significant differences result from two-population t tests and one-way ANOVA test . Table 2Statistics for tensile parameters of ape hairs FEαFrSrGibbon × Orangutant1011188 .44.4 p < 0.0001 < 0.0001 < 0.00010.0003 Gorilla × Gibbont105 .36.24.10.32 p < 0.0001 < 0.00010.00080.75 Gorilla × Orangutant103 .17.61.34.0 p0 .007 < 0.00010.20.0009 ANOVAF2 ,2744119209.3 p < 0.0001 < 0.0001 < 0.00010.0009 Results from statistical tests for the n = 10 measured values of the four parameters obtained from the force × strain curves of hairs of the three apes . Two-population t-test -LRB- t10 values and corresponding probability p -RRB- are given for each parameter and each compared population in the upper lines . ANOVA test of the four parameters in the three ape populations and corresponding probability p in the lower line Hominoid species differ significantly regarding hair strength , which increases from gibbon to gorilla and to orangutan . However , the Young modulus has a different behavior : E values for gibbon and orangutan are equal -LRB- 2.8 Gpa -RRB- , but gorilla has a higher value -LRB- 5.0 Gpa -RRB- . The value for human head hair from literature -LRB- Robbins 1994 ; Nikiforidis et al. 1993 -RRB- is 3.8 Gpa , intermediate between gorilla and orangutan hair . Human head hairs measured in this paper are within reported values , as detailed in the Appendix . Results obtained indicate that the force strength and average thickness of hominoid hairs are species-dependent and may be evolutionary meaningful . Data for human hair in the Appendix give further indication of species dependence . Such direction of research , together with definition of the Young modulus in terms of the inner hair structure , have clear interest but outside the scope of this work . For infant carrying , the really decisive parameter is FE , representative of the upper limit of the force that a single hair can safely withstand , because it is the limit for recovery after relaxing . It is convenient to express FE in kilogram-force -LRB- and gram-force -RRB- , corresponding to the force weight of a given mass at the earth surface . This unit is often still used for load -LRB- instead of N = 0.10198 kgf -RRB- because it makes the mass value of the weight clear . The important values FE are then 18 ± 2 gf for gibbon , 39 ± 3 gf for gorilla , and 50 ± 2 gf for orangutan . The value for human head hair is 29 ± 2 gf -LRB- see Appendix -RRB- . The force to pull hairs from the skin samples of the three ape species has also been measured and may be considered as an upper limit , in view of changes in skin due to taxidermy . They are typically near the elastic limit and lower than rupture value , as occurs with human head hair in the scalp -LRB- Robbins 1994 -RRB- , giving a further reason to focus on the FE values as upper limits for safe load carrying . A robust conclusion can be thus extracted . It is clear that to withstand the clinging infant weight , a large number of hairs held together is required . Results obtained evidence that bunches of about 100 hairs are necessary to carry infants weighing a few kilogram-force . Infant hands -LRB- and feet -RRB- can possibly grasp a bunch of hairs available in some square centimeters of skin so that safety in the usual pattern of nonhuman primate infant carrying critically depends on the density of hairs . Infant carrying Values of hair density for nonhuman primates have been obtained in the extensive work of Schultz -LRB- 1931 , 1968 -RRB- ; values are not gender-dependent but vary with body region : density is highest in the vertex and smaller on the ventral than on the dorsal side of the trunk . Densities are generally compatible with the carrying requirement defined above , but the great apes have the lowest hair density among primates . Hair density values on the back of primates vary , from over 1,000 hairs/cm2 for smaller species -LRB- monkeys and gibbons -RRB- , down to about 100 hairs/cm2 for great apes . The problem of safely carrying heavy infants requires thus particular consideration among the great apes . Analysis of hair density data -LRB- Schultz 1931 -RRB- in many anthropoid primate taxa yield -LRB- Schwartz and Rosenblum 1981 -RRB- a negative allometry of relative hair density -LRB- rhd = hair density/total surface area -RRB- . This decrease in rhd with increase in primate body mass has been correlated to thermal constraints imposed by the decreasing ratios of surface area to body volume . However , chimpanzees apparently have a lower rhd than would be expected for their body volume -LRB- see Fig. 1 of Schwartz and Rosenblum 1981 -RRB- , with rhd similar to that of larger gorillas . The bunch of hairs available for clinging depends also on hair length and infant hand size . Accepting that about 3 cm are necessary to roll hairs on the infant fingers , the excess in length defines the area available for clinging . Clinging area per limb may be thus estimated to be 3 , 10 , and 50 cm2 for , respectively , gibbon , gorilla , and orangutan . In a dynamic situation , the load should be supported instantaneously by a single limb . The infant-carrying method of the arboreal Asian apes -LRB- gibbon and orangutan -RRB- is different from that of the terrestrial African apes -LRB- gorilla and chimpanzee -RRB- . The arboreal apes carry their young over one side of the mothers ' pelvis , not on the back -LRB- de Vore 1965 -RRB- . In accordance with the small size -LRB- 7 kgf when adults -RRB- , the lesser ape gibbon has a high hair density , over 1,000 hairs/cm2 , more than ten times the density of great apes -LRB- Schultz 1931 , 1968 -RRB- . This ensures safety for carrying their light young in their acrobatic arboreal life . The arboreal but heavier orangutans -LRB- adult females weigh around 50 kgf and males 90 kgf -RRB- compensate the lower hair density by longer , thicker , and stronger hairs and thicker skin . Orangutan infants live with their mothers for about 7 years . The gorilla is about 50 % heavier than orangutan , but instead of even greater hair thickness and length , the solution for terrestrial apes is the mounting position for heavier infant support . Orangutan hair and skin are , in this paper , estimated to be able to withstand about seven times more weight than gorilla hair and skin . Gorillas are the heaviest primates and have very few chest hairs -LRB- Schultz 1931 , 1968 -RRB- . Adult gorilla is much heavier than adult human , but their newborns have only half the weight of human babies . In the first 1 or 2 months , the infant gorilla is supported manually by its mother as she walks tripedally or bipedally -LRB- Jolly 1972 ; de Vore 1965 ; Tuttle and Watts 1985 ; Doran 1997 -RRB- . The same occurs for chimpanzee babies , unable to support their own weight by clinging prior to 2 months of age -LRB- Plooij 1984 -RRB- . Change from quadruped to triped or biped motion occurs systematically among all great apes when infant safety requires manual support -LRB- Jolly 1972 ; de Vore 1965 ; Tuttle and Watts 1985 -RRB- . Slow and careful locomotion of female chimpanzees while carrying young infants has been reported -LRB- Goodall 1967 -RRB- . Two primary causes of mortality have been found among infant chimpanzees : inadequacy of the mother -- infant bond and injuries caused by falling from the mother -LRB- Goodall 1967 -RRB- . It is clear that infant carrying is crucial and depends on not trivial behavior among African great apes . Weight limit for clinging in African apes The gorilla FE value , combined with hair density and length , indicates that load up to 40 kgf could be supported by clinging . However , the weight limit for clinging depends also on several other variables . Increase in hair pull-out or break in the dynamic situation , capacity of the infant to withstand its own weight and to grasp hairs without sliding , and skin capacity to withstand the load without rupture are all variables that may decrease the infant weight limit for clinging . A bunch of hairs on about 1 cm2 -LRB- ± 10 % -RRB- of gorilla skin under load was observed , still on the skin , to simulate the real load effect in the actual clinging situation . Counting hairs in this bunch gave 140 hairs -LRB- ± 10 % -RRB- , in agreement with the value reported by Schultz -LRB- 1968 -RRB- for the back side of the trunk of gorillas -LRB- 145 hairs/cm2 -RRB- , evidencing that there are no marked differences between the limb sample and the back-trunk hairs . This bunch of hairs supported 1 kg without problems , even with some pendulum movement . With 2 kg , several problems became evident . Besides eventual detachment of single hairs from the skin during movement , the hair bunch started to slip from the double-face adhesive tape used to attach it to the load support . It was necessary to make a new attachment system ; the hair bunch was rolled several times around the weight support . Even so , loads of 2 kg and more could be observed only with the rolled hairs attached by super glue -LRB- ester cyanoacrylate -RRB- to the weight support . This indicates a possible problem also with the clinging capacity of infant fingers , and slippage may occur . Also , the skin itself began to deform and detach from the wood support , requiring a much stronger attachment of the skin to the wood base . The bunch of hairs broke at the same hair length for all under a static load of 7 kg , compatible with the values of force at rupture -LRB- Fr -RRB- obtained from the tensile measurements of single hairs -LRB- Table 1 -RRB- . An upper limit of 1 kgf/cm2 for clinging without problems may be deduced from this simple experiment , but the actual limit depends on the clinging capacity of infant fingers and on the mother 's skin resistance . The effective weight limit for clinging must be obtained from careful observation of live apes . Fossey -LRB- 1979 -RRB- describes physical and behavioral development of gorilla infants : at 4 -- 6 months -LRB- weight about 5 kgf -RRB- , the gorilla infant travels 60 % of the time in ventral position while with 6 -- 12 months -LRB- weight about 8 kgf -RRB- , it travels in the dorsal position 80 % of the time . A detailed investigation of the ontogeny of locomotion in the African ape -LRB- Doran 1997 -RRB- revealed that up to 5 months of age , chimpanzees are slightly more precocious than gorillas . However , at 6 months , gorilla locomotor development becomes faster than that of chimpanzees , and surprisingly , much of the interspecific variation in behavior is explained by differences in body size -LRB- Doran 1997 -RRB- . Afterward , when the infants are of similar weights -LRB- although of widely disparate ages -RRB- gorillas and chimpanzees perform very similar locomotion activities -LRB- Doran 1997 -RRB- . It is , in this paper , suggested that the weight level of 5 kgf may be considered the limiting value for safe support over long periods by only ventral clinging , defined probably by a combination of security factors1 related to hair strength , infant clinging capacity , and skin resistance to pressure . The limiting clinging pressure on the mother 's skin for the African great apes may be thus estimated to be 0.5 kgf/cm2 -LRB- a factor of 2 in relation to the simple test made with the skin sample -RRB- . The skin is also a visco-elastic medium , reflecting properties of a strain-induced aligned-collagen network that can stretch up to 100 % before permanent damage -LRB- Silver 1987 -RRB- . The shear modulus for the human dermis ranges from 1.2 to 3.1 MPa , while for the human hypodermis , it ranges from 3.1 to 9.7 kPa -LRB- Gennisson et al. 2004 -RRB- . The pressure limit 0.5 kgf/cm2 -LRB- ~ 50 kPa -RRB- could be a limit also for danger to the skin of the mother ape . It is known that one of the important differences between the skin of humans and of African apes is the higher elasticity of the human skin -LRB- Montagna 1982 -RRB- . The locomotor development of the infants must therefore adapt to such an effective weight limit . This does not mean that infants over 5 kg will fall . Larger infants may travel briefly in ventral position during stressful situations -LRB- Fossey 1979 -RRB- or when the chimpanzee mother swings through trees -LRB- Goodall 1967 -RRB- . Usual locomotion of adult African apes is terrestrial quadrupedalism -LRB- ~ 96 % of the time in gorillas and ~ 86 % in chimpanzees , from Doran 1997 -RRB- , and safe infant support over longer periods clearly requires the necessity of change to a mounting position , with the infant weight supported by the adult body . Dorsal clinging position In order to analyze the mechanics of the carrying system in the African great apes , it is also necessary to consider , besides the tensile properties of hairs , the hair -- hair friction that prevents slipping , particularly for heavier infants in dorsal position . Fig. 3 is a sketch of the mounting position in African apes , showing the angle θ of the inclined plane where the infant stands . For simplicity in the sketch , the angle θ coincides with the angle defined by the usual knuckle-walking position of African great apes , but that is not necessary . The important parameter is the inclination of the base where the infant stands clinging ; its relation to the average body inclination angle does not need to be taken into account in analysis of the friction effect . Fig. 3Sketch of ape carrying infant . For simplicity , in the sketch , the angle θ of the inclined plane where the infant stands -LRB- to which Eq . 1 refers -RRB- coincides with the angle defined by the knuckle-walking position of African great apes , which is not necessarily so , as discussed in the text The total infant weight , in the vertical direction , has two components , one in the inclined plane , favoring slipping , and one normal to the inclined plane , responsible for the friction force opposing slipping . Calling Wt the total infant weight carried in mounting position , Wc the effective weight limit for clinging , and μ the static friction coefficient -LRB- Bowden and Tabor 1956 -RRB- , the equilibrium of forces on the infant requires that force components on the inclined plane direction satisfy the condition : In the absence of clinging -LRB- Wc = 0 -RRB- , slipping on the body surface of contact starts for a critical value θc , given by tan θc = μ . Both friction and clinging are thus essential to hold heavy infants in the mounting position . The requirements for dynamic equilibrium may be greater , so that in fact the mounting position in dynamic movement may be unsafe even when Eq . 1 is satisfied . It should be stressed that Eq . 1 is a necessary condition for infant survival and therefore also for species survival . It is a very robust requirement of basic mechanics for static equilibrium . The ape hair -- hair friction coefficient μ must be known to analyze the actual ape situation . Friction measurements Apparently , no results for hair -- hair friction coefficients exist in the literature ; only friction between human hair and other materials are reported -LRB- Robbins 1994 -RRB- . The skin samples were used to measure μ as a function of the supported load W in known conditions of relative air humidity and temperature , in a situation analogous to that depicted in Fig. 3 . It was verified that μ depends on the hair direction , being larger for hairs in the parallel position -LRB- as in nature , with the two animals heading in the same direction -RRB- than in the antiparallel condition . Systematic measurements have been thus made in the parallel condition . Figure 4a shows a typical result of μ as a function of W. For hard solid surfaces , μ is constant and independent of the load W and contact area A . This is clearly not the case for hairs , especially at low loads . Long ago , it was shown that a variation of μ with W occurs for textiles and fibers -LRB- Gralen 1952 ; Makinson 1952 -RRB- , given by the empirical equation : Fig. 4Results for friction coefficient at 80 % RH . a friction coefficient μ as a function of W for a gorilla skin with smaller area -LRB- 16 cm2 -RRB- . The line shows a fit to the data using Eq . 2 . bμ × W for -LRB- circles -RRB- gorilla with larger area -LRB- 76 cm2 -RRB- ; -LRB- squares -RRB- gorilla with smaller area -LRB- 16 cm2 -RRB- , and -LRB- triangles -RRB- gibbon with intermediate area -LRB- 42 cm2 -RRB- . Linear fits to the data are shown in lines The parameter a gives the friction coefficient in the limit of high loads . It has been proposed -LRB- Gralen 1952 -RRB- that the parameter b is related to the pressure inside an oil lubricating film on the fiber surface . The thickness of this film decreases due to pressure , which causes changes in the friction mechanism . Hair is a natural fiber -LRB- Howell et al. 1959 -RRB- and behaves in a similar way . In fact , the data could be well-fit with Eq . 2 , shown as a solid line in Fig. 4a . Many curves were analyzed for different values of the contact area A and RH . A strong dependence of μ on A was observed , probably due to shear among the several hairs in contact . The fits to the linear equation μW = aW + b are more convenient for yielding the parameters a and b , as shown in Fig. 4b . Both a and b increase with A , but a is less sensitive to A , while b is roughly proportional to A , so that the term b/W in Eq . 1 is inversely proportional to the pressure W/A exerted on the surfaces in contact . Results indicate that the friction coefficient might be considered constant for pressures higher than 20 gf/cm2 . This pressure corresponds to infant weight around 1 kgf , from the relation between body surface area and weight -LRB- Schwartz and Rosenblum 1981 ; Kleiber 1975 -RRB- and estimating the contact area as one third of the infant surface area . The average value is μ = 0.27 ± 0.03 for gorilla hairs and μ = 0.20 ± 0.02 for gibbon hairs at 60 % RH , in agreement with the expected null or small difference on friction depending on the fiber diameter -LRB- Robbins 1994 -RRB- . Such values are in good agreement with known values for single fibers -LRB- Bowden and Tabor 1956 ; Howell et al. 1959 -RRB- . The orangutan skin was not as perfect and homogeneous as the gibbon and gorilla skins , showing some parts without hairs , and provided only two samples , so dependence on area could not be analyzed . However , experiments of μ as a function of load gave similar values to those for the other apes . The hair -- hair friction coefficient of apes obtained is intermediate between values for friction between human hair and hard rubber with dry -LRB- μ = 0.19 -RRB- and wet -LRB- μ = 0.38 -RRB- surface conditions -LRB- Robbins 1994 -RRB- , indicating that results obtained can be considered of general validity . Note that Robbins -LRB- 1994 -RRB- mentions the difficulty of testing hair -- hair friction , important in hair combing , and that it should be similar to hair -- rubber friction . Equilibrium condition in African apes It is possible now to focus the problem on our nearest relatives , the great apes gorilla and chimpanzee , applying Eq . 1 to their mounting condition , in which clinging and friction act together for safe infant carrying . The effective weight limit for constant ventral clinging , already discussed , is estimated to be 5 kgf . As the hair density in the back is higher -LRB- 30 % for chimpanzees -RRB- , the estimate is Wc ~ 5 -- 7 kgf . The maximum value Wt can be estimated from the weight of infants when they are no longer carried by mothers . This occurs when gorillas are about 2.5 years old and chimpanzees 5 years old , both weighing around 20 kgf -LRB- Fossey 1979 ; Doran 1997 -RRB- . By inserting such values in Eq . 1 , and using the estimated constant friction coefficient μ , it is possible to obtain the maximum inclination of the plane supporting the infant for the safe carrying system in the African great apes . The condition θ < 29 to 34 ° is obtained . This basic equilibrium constraint related to friction in the mother -- infant contact is satisfied in ape knuckle-walking quadruped position2 . It should be stated that it is frequently reported that in the dorsal position , the African ape infant rides high on the mother 's neck and lies stretched and prostate -LRB- de Vore 1965 ; Fossey 1979 -RRB- . This position increases the fixed base of the load and places it in a body region with a promontory and a smaller inclination angle -LRB- θ ~ 20 ° as estimated2 from the same Fig. 7 -- 20 in Schultz 1968 -RRB- . Older infants ride in a jockey position ; they do not normally grip hairs with their feet , but press the sides of their ankles against the mother 's flank -LRB- Goodall 1967 -RRB- . Clearly , among terrestrial great apes , infant safety does not rely only in grasping hairs . It may be concluded that slipping imposes clear limits on the maximum body angle attained by heavy ape species carrying infants , representing a hindrance against evolution towards bipedality . This might explain the persistence of knuckle-walking among the great African apes . Effect of humidity on friction The two most relevant variables to friction in hairs -LRB- Robbins 1994 -RRB- are the load pressing the two surfaces together , as analyzed previously , and the relative humidity RH . Dependence of μ on RH was reported for fibers -LRB- Howell et al. 1959 -RRB- and human hair -LRB- Robbins 1994 -RRB- , and a similar effect was verified for ape hairs . Reduction in RH , from 80 to 38 % , led to a decrease of gorilla hair μ by about 30 % . The friction coefficient between human hair and hard rubber is reduced by 50 % between the wet and dry states -LRB- Robbins 1994 -RRB- . Friction of gorilla hairs has been also measured in wet conditions -LRB- immersion in water , followed by slight shaking of the sample -RRB- . The result is μ = 0.35 ± 0.03 , in good agreement with reported values for human hair and hard rubber in the wet condition . The larger μ value in wet condition is possibly due to network of hydrogen bonds connecting hairs in contact . The thin outer epicuticle membrane of hair is hydrophobic , but hydrogen bonds may occur through the thicker exocuticle and endocuticle , especially the latter , which swells much more in water -LRB- Feughelman 1997 , Robbins 1994 -RRB- . Such structures may be accessible to external water through the ratchet scale structure of hair . It should be concluded that reduction in humidity destroys the delicate balance between friction and clinging -LRB- Eq . 1 -RRB- necessary for survival of infants of heavy ape species . Figure 5 shows the maximum carrying weight Wt obtained from Eq . 1 as a function of the inclination angle θ for μ varying from zero up to 0.40 , with Wc = 5 kgf . Fig. 5Curves of total weight Wt supported in dorsal position as function of the inclination angle θ of the infant base on the mother 's trunk , obtained from Eq . 1 , for several values of the friction coefficient μ -LRB- 0 -- 0.4 , as indicated on top -RRB- , with Wc = 5 kgf -LRB- limit value shown with dashed line -RRB- . The line with cross is for Wc = 10 kgf and μ = 0.4 . Vertical position corresponds to θ = 90 ° Discussion In this section , the relevance of the above results for models of hominization is discussed . Safe infant carrying is clearly very important for species survival and has to be considered in the discussion of the transition from apes to hominins . The mechanical analysis made in this paper evidenced that habitual bipedalism , with a large θ angle , is incompatible with the usual primate type of infant carrying . Even if one considers Wc = 10 kgf and μ = 0.40 , the maximum attainable angle is θ ~ 49 ° , as seen in Fig. 5 . It seems clear that together with bipedality probably also came the characteristic human type of infant carrying in the arms and hands of their mothers , making their locomotion and foraging particularly difficult . Females would not adopt bipedality if they could avoid it , and the species would not adopt bipedalism if females with infants would not engage in it . The transition to bipedality was not trivial . Etkin -LRB- 1954 -RRB- considered the problem of the burden on females `` almost continuously carrying a child , '' just to conclude that the female could not be an effective hunter , turning to a theory of the monogamous family unit . It is clear now that such a scenario could be considered only for Homo at a much later period . Iwamoto -LRB- 1985 -RRB- recalled examples of facultative bipedalism among monkeys and speculated that the `` decisive factor -LRB- for habitual bipedalism -RRB- may have been some everyday necessity to carry something in both hands '' . After criticizing proposals in which the `` something '' is food , Iwamoto suggested that the `` something '' could be their helpless babies . However , this proposal has been dismissed under the hypothesis that babies became helpless only with increase of brain in Homo . Now models for bipedalism origin based on `` food acquisition and foraging strategies '' are considered `` likely '' but are in fact based on the premises that bipedal locomotion frees the arms and hands and does not have high costs . However , such premises just forget the fundamental fact that bipedalism frees the arms and hands only of males and juveniles , but females , on the contrary , have arms and hands occupied by infant carrying , which represents a high cost for bipedalism , not considered up to now . A review of bipedalism mentions : `` infants -LRB- from early hominin biped -RRB- probably clung to their mothers ' hair , as is the case for most other primates '' -LRB- Richmond et al. 2001 -RRB- . It seems that the not trivial mechanical constraint of safe carrying of heavy infants has never been considered before . Current evidence suggests -LRB- Richmond et al. 2001 ; Kingston et al. 1994 ; Potts 1998 -RRB- that the key adaptation to bipedalism originated in wooded environments during a drier period in East Africa . The equilibrium condition -LRB- Eq . 1 -RRB- shows that reduction of friction in the dorsal clinging position would most probably lead to a decrease in infant weight and decrease in the body inclination angle θ , corresponding to the solution adopted by quadruped savanna monkeys in the arid savanna . It seems clear that habitual bipedalism would not be reached by continuous increase of the body angle . However , among hominoids , reduction of friction between infant fingers and mother hairs could reduce infant grasping capacity , increasing the period of mother 's manual support of newborns and eventually inducing habitual bipedalism . Concluding remarks and future perspectives An important conclusion from this work refers to interest in systematic study of tensile properties of hairs from an evolutionary point of view . The other clear conclusion is that models on locomotion and bipedalism evolution must focus on females carrying infants , who are the target of the strongest selective pressures , and this conclusion is independent of the form of locomotion of hominid ancestors -LRB- knuckle-walkers , terrestrial or arboreal quadrupeds , or full-time arboreal climbers -RRB- . To disentangle the divergence between apes and hominins , several directions of research become clear from this work . Studies of living apes focused on the mechanical problem of infant carrying , as well as determination of the elastic limits of the ape skin and the limits for grasping capacity of ape infants , which are all essential to identify the critical factor responsible for emergence of bipedalism . It should be stressed that in a scenario with bipedalism emerging for safe infant carrying , the selective pressure would act particularly on females . This is not a problem , as only genes in the nonrecombinant part of the Y chromosome are not transmitted to both sexes and therefore can not be associated with species locomotion . The process correlates with increase in the mother -- infant bond , characteristic of the human lineage . Other hypotheses for emergence of bipedality -LRB- see Richmond et al. 2001 for a review -RRB- do not , in fact , explain why other primates did not follow such line . The many scenarios invoked previously for bipedalism evolution may sound plausible , but are not necessary ; neither take into account the problem of infant carrying and can be considered complementary and a consequence of the selective pressure on females carrying infants . The difficulties in locomotion and food gathering for biped females carrying infants may well be at the origin of the necessity of group cooperation , which could initially have been among females , with males in their usual role of primate group protection . A fundamental question is the possible correlation between bipedalism and reduction of body hairs , the two basic biological modifications of hominins . It is evident that decrease in body hair as the initial modification would bring on bipedality as a necessary consequence -LRB- Amaral 1989 -RRB- , through the strong selective pressure of safe infant carrying , when infants could no longer cling to body hairs . The timing of reduction of body hairs is very controversial , since Darwin 's original -LRB- 1871 -RRB- emphasis on the issue . Several works indicated a very early beginning of body hair reduction , still in forested environments , based on thermoregulatory requirements -LRB- Newman 1970 -RRB- , rhd of Australopithecines -LRB- Schwartz and Rosenblum 1981 -RRB- , and comparison of human and ape skin -LRB- Montagna 1982 , 1985 -RRB- . Bipedality preadaptive to nakedness was suggested later from water-consumption requirements under reduced humidity -LRB- Wheeler 1992 -RRB- . However , detailed analysis of thermal loads and water consumption for quadrupeds and bipeds in the furred and naked conditions suggested again that reduction of body hair started in a forested environment becoming drier -LRB- Amaral 1996 -RRB- . Savanna monkeys tolerate heat even at high levels of exercise , evidencing that the biological avenue to cope with heat stress in an open environment is to keep a hair covering , increase sweating capacity , and have a variable conductance -LRB- patas monkeys , Mahoney 1980 , and baboons , Rogers et al. 1992 -RRB- . The only situation in which nakedness is favorable concerns dissipation of heat loads that do not come from sun absorption but from activity in a more closed forest condition at temperatures below that of the body -LRB- Amaral 1996 -RRB- . Furthermore , regarding water requirements , advantages for nakedness exist in dry ambients with temperatures below body temperature -LRB- Amaral 1996 -RRB- . Recent genetic work has focused on evolution of black skin as a result of naked unprotected skin under solar stress , estimated to have occurred at least 1.2 mya -LRB- Rogers et al. 2004 -RRB- , while clothing is a recent innovation of only about 70,000 years ago -LRB- Kittler et al. 2003 -RRB- . However , up to now , there is no date for the beginning of reduction of body hairs . Therefore , localization of the genetic changes responsible for reduction of body hairs in humans is necessary to settle the issue . The new perspective developed in this paper evidences continuity between physical and behavioral aspects of early hominins and their ancestors . The more differentiated aspects of humans are left to the emergence of Homo , at a much later period .

O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B I O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O B I O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

infant carrying
hair strength
clinging
friction
bipedalism
reduction of body hairs


TbARF1 influences lysosomal function but not endocytosis in procyclic stage Trypanosoma brucei The ADP ribosylation factors -LRB- Arfs -RRB- are a highly conserved subfamily of the Ras small GTPases with crucial roles in vesicle budding and membrane trafficking . Unlike in other eukaryotes , the orthologue of Arf1 in the host bloodstream form of Trypanosoma brucei is essential for the maintenance of endocytosis . In contrast , as shown in this study , knockdown of TbARF1 by RNA interference has no effect on fluid-phase endocytosis in the insect stage of the parasite . The protein remains essential for the viability of these procyclic cells but the major effect of TbARF1-depletion is enlargement of the lysosome . Our data indicate that protein trafficking and lysosomal function are differentially regulated by multiple factors , including TbARF1 , during progression through the T. brucei lifecycle . 1 Introduction The secretory system plays a critical role in the efficient sorting and transport of a range of products to the surface of all eukaryotic cells . The small GTPase Arf1 is a vital component of this system , localizing to the Golgi apparatus in yeast and mammalian systems where it has highly conserved functions in vesicular assembly and the activation of phospholipid-modifying enzymes -LSB- 1 -RSB- . An exception is the orthologue of Arf1 -LRB- TbARF1 -RRB- that has been recently characterized functionally in the bloodstream form -LRB- BSF -RRB- of the kinetoplastid parasite Trypanosoma brucei -LSB- 2 -RSB- , a cell type which supports extremely rapid rates of internalisation and recycling of its major outer membrane protein , variant surface glycoprotein -LRB- VSG ; -LSB- 3 -RSB- -RRB- . Although TbARF1 localizes at or proximal to the Golgi apparatus in these parasites , the primary effect of TbARF1 knockdown is severe inhibition of the endocytic system , leading to cell death . In contrast , overexpression of a constitutively active GTP-bound form of TbARF1 inhibits protein trafficking from the Golgi apparatus to the lysosome -LSB- 2 -RSB- . Here we show that the vital role of TbARF1 in endocytosis is not conserved throughout the T. brucei lifecycle . In the more sedentary insect procyclic form -LRB- PCF -RRB- of the parasite , ARF1 is required for cell viability but depletion of the protein by RNA interference -LRB- RNAi -RRB- has no effect on the uptake of dextran by fluid-phase endocytosis . Instead , the lysosome becomes enlarged , although degradation of the protein p67 within this organelle is not significantly impaired . 2 Materials and methods 2.1 Disruption of TbARF1 expression by RNAi The plasmid p2T7ARF1 -LSB- 2 -RSB- contains a non-conserved region of the T. brucei open reading frame -LRB- residues 101 -- 225 -RRB- between two opposing T7 promoters under the control of tetracycline repressors . Mid-log phase parasites of T. brucei procyclic cell line 29 -- 13 -LSB- 4 -RSB- were electroporated with 10 μg of NotI-digested p2T7ARF1 using methods described previously -LSB- 5 -RSB- to produce the stable cell line 29-13 / p2T7ARF1 . Expression of ARF1-specific dsRNA was induced by incubating parasites in 100 ng/ml tetracycline . Expression of TbARF1 was monitored by quantitative PCR , using SYBR Green Mastermix -LRB- Applied Biosystems -RRB- . Total RNA was extracted from parasites using TRIzol Reagent -LRB- Invitrogen -RRB- , treated with DNase I -LRB- Ambion -RRB- and reverse-transcribed using Omniscript RT -LRB- Qiagen -RRB- . A 66 bp fragment of TbARF1 was amplified using SYBR Green Mastermix -LRB- Applied Biosystems -RRB- on a Prism7000 -LRB- Applied Biosystems -RRB- and compared to levels of a constitutively expressed control , α-tubulin . Oligonucleotides for amplification were : TbARF1 RTF1 , 5 ′ - GGCTTCCGCTTTCAAATCC-3 ′ , TbARF1 RTR1 , 5 ′ - CATCAAGGCCGACCATAAGAA-3 ′ , TbαTub RTF1 , 5 ′ - CGTGAGGCTATCTGCATCCA-3 ′ and TbαTub RTR1 , 5 ′ - CCCAGCAGGCGTTACCAA-3 ′ . 2.2 Microscopy Indirect immunofluorescence assays on parasites were performed as described -LSB- 6 -RSB- . Primary antibodies were gifts as follows : rabbit anti-TbGRASP from Graham Warren -LRB- Department of Cell Biology , Yale University School of Medicine , New Haven , CT , USA -RRB- , mouse anti-p67 from Jay Bangs -LRB- Department of Medical Microbiology and Immunology , Madison , WI , USA -RRB- . Primary antibodies were detected using Alexa-Fluor 488 or 633-conjugated secondary antibodies -LRB- Invitrogen -RRB- . Samples were visualized by confocal microscopy using a Zeiss LSM 510 meta with a Plan-Apochromat 63x/1 .4 Oil DIC I objective lens and images acquired using LSM 510 version 3.2 software -LRB- Zeiss -RRB- . For transmission electron microscopy , cells were sequentially treated in 1 % -LRB- w/v -RRB- glutaraldehyde for 1 h , 1 % -LRB- w/v -RRB- tannic acid for 10 min , 0.5 % -LRB- w/v -RRB- osmium tetroxide for 45 min -LRB- all in 100 mM phosphate buffer -RRB- , then in 1 % -LRB- w/v -RRB- aqueous uranyl acetate for 1 h . After dehydration in an acetone series , cells were embedded in Spurrs resin . Sections were cut on a Leica Ultracut , stained with saturated uranyl acetate in 50 % ethanol and Reynolds lead citrate , and viewed with a Tecnai 12 BioTwin -LRB- FEI -RRB- at 120 kV . Images were acquired with a SIS MegaView III digital camera . 2.3 Trafficking assays To monitor the uptake of dextran , cells were resuspended at 5 × 107/ml in 1 mg/ml Alexa-Fluor 488-conjugated dextran -LRB- Invitrogen -RRB- in SDM-79 medium and incubated for 20 min at 26 °C . For microscopy , parasites were then fixed in 4 % paraformaldehyde and co-stained with DAPI , as described previously -LSB- 7 -RSB- . For FACS analysis , cells were fixed as above , then resuspended in PBS at a density of 5 × 107/ml . Samples were then divided into two equal aliquots and anti-488 antibody -LRB- 1:100 , Invitrogen -RRB- added to one aliquot for 30 min at RT , before washing in PBS . Fluorescence was measured on a Dako Cytomation CyAn flow cytometer using the FL1 detector and results analyzed with Summit v4 .1 software . ER-to-lysosome trafficking and subsequent degradation of the protein p67 was analyzed by metabolic labelling of cells and immunoprecipitation as described previously -LSB- 8 -RSB- . 3 Results 3.1 TbARF1 is essential for viability in T. brucei procyclic cells We studied the functions of TbARF1 in PCF cells by knocking down expression using tetracycline-inducible RNAi . Detection of phenotypic effects arising from TbARF1 knockdown in these cells was considerably delayed in comparison to BSF parasites -LSB- 2 -RSB- in which cell death was evident by 24 h post-induction . This probably reflects a lower dependence on rapid protein trafficking in the PCF cell , as observed previously -LSB- 9 -RSB- . There was no significant decrease in PCF cell division until 90 h post-induction , when the cells started to become rounded and less motile , followed by cell death from 96 h -LRB- Fig. 1A , B -RRB- . Quantitative PCR showed a significant loss of TbARF1-specific RNA by 16 h post-induction -LRB- Fig. 1C -RRB- . Induced cells had no significant differences in nucleus and kinetoplast configurations between uninduced and induced cells over a 120 h time course -LRB- data not shown -RRB- , indicating no major defects in the regulation of cell division . 3.2 Loss of ARF1 has no effect on fluid-phase endocytosis The effects on fluid-phase endocytosis in these TbARF1-depleted procyclic cells were determined by analyzing the uptake of fluorescently labelled dextran . Analysis by microscopy showed internalization of dextran in both uninduced parasites and cells induced with tetracycline for 96 h -LRB- data not shown -RRB- . We also used a novel FACS-based method to distinguish between external binding and internalization of dextran in these cells -LRB- Fig. 2A -RRB- . Cells were incubated in the presence of Alexa Fluor 488-conjugated dextran , before fixing and incubation in anti-488 antibody which quenches the fluorescence associated with externally bound dextran . No significance differences were observed between uninduced and induced cells , either in total dextran fluorescence or internalized dextran alone -LRB- i.e. following antibody treatment -RRB- -LRB- Fig. 2A -RRB- . 3.3 Loss of ARF1 causes an enlargement of the lysosome Immunofluorescence assays using the lysosomal marker , p67 , showed that TbARF1-depleted procyclic parasites have an enlargement of the lysosome -LRB- Fig. 2B -RRB- . Densitometric analysis of 20 cells per experimental group revealed that p67 staining in cells induced for 96 h covered an area approximately three times as large as in uninduced cells . No differences were seen in the size or position of the Golgi apparatus , as indicated by localization of the GRASP protein -LRB- Fig. 2B -RRB- . The main defect visualized by transmission electron microscopy was the presence of a single electron-dense rosette-like structure near the flagellar pocket in a subset -LRB- 30 % -RRB- of cells by 72 h post-induction , before detection of a growth defect in these cells . This structure -LRB- Fig. 2C -- E -RRB- is composed of concentric layers of membrane and resembles that of membranous cytoplasmic bodies -LRB- MCBs -RRB- found in mammalian cells . These are lysosomal in origin and typically found in sphingolipidoses , a group of lysosomal storage diseases characterized by deficiencies in the lysosomal enzymes required for sphingolipid degradation -LSB- 10 -RSB- . Similar structures of mitochondrial origin have also been observed in T. brucei following depletion of the glycosomal PEX11-like protein GIM5B -LSB- 11 -RSB- . However , we found no obvious differences in mitochondrial structure in procyclic cells following the induction of TbARF1 RNAi , either by electron microscopy or fluorescent staining with Mitotracker -LRB- Invitrogen , data not shown -RRB- . We therefore conclude that the structure detected in Fig. 2 is lysosomal in origin , although proof of this designation requires immuno-electron microscopy with a lysosomal probe . 3.4 Effects of ARF1 depletion on lysosomal trafficking and degradation of p67 The trafficking and degradation of p67 was analyzed by pulse-chase metabolic labelling of cells , followed by immunoprecipitation , as described previously -LSB- 5,8 -RSB- . In contrast to this process in BSF parasites , ER - synthesized p67 is not further modified in the Golgi in PCF cells , but proteolytic cleavage in the lysosome results in the generation of four glycosylated fragments : gp75 , gp42 , gp32 and gp28 -LSB- 8 -RSB- . In Fig. 2F , proteolytic fragments resulting from lysosomal degradation accumulated in uninduced and induced cells over the same time course , indicating that both trafficking of p67 and its subsequent degradation were unimpaired in cells depleted of TbARF1 protein -LRB- Fig. 2F -RRB- . 4 Discussion The observations presented here and described previously -LSB- 2 -RSB- show that TbARF1 is essential for viability throughout the T. brucei lifecycle . However , the downstream effects of modulating expression of this protein differ between the two major life cycle stages . In the highly active bloodstream form , TbARF1 is required for both receptor-mediated and fluid-phase endocytosis , and depletion of the protein by RNAi causes a severe defect in these mechanisms , rapidly followed by cell death -LSB- 2 -RSB- . In contrast , procyclic cells significantly downregulate the expression of proteins associated with receptor-mediated endocytosis , including clathrin -LSB- 12 -RSB- , and fluid-phase endocytosis is unaffected by the knockdown of TbARF1 expression . Rather than affecting the endocytic system , the absence of TbARF1 causes enlargement of the lysosome , indicating an accumulation of undegraded and/or mistargeted material . An interesting finding is that lysosomal targeting of p67 is not disrupted by knockdown of TbARF1 in either of the two life cycle stages but is inhibited by an increase in activated ARF1 protein in BSF parasites -LSB- 2 -RSB- . Lysosomal targeting in trypanosomes is not yet fully understood but must differ from higher eukaryotes in several respects . In mammals , most soluble lysosomal enzymes , including acid hydrolases , are sorted using mannose-6-phosphate signals -LSB- 13 -RSB- but there is no evidence to suggest that this mechanism is conserved in lower eukaryotes such as the kinetoplastids -LSB- 8 -RSB- . An alternative to the mannose-6 receptor , sortilin , has also been implicated in hydrolase sorting -LSB- 13 -RSB- but orthologues of this protein are not encoded by the T. brucei genome . The remaining class of identified signals , either tyrosine or dileucine-based motifs , are able to bind to GGA and AP adapter proteins , which then trigger the packaging of cargo for clathrin-mediated trafficking from the trans-Golgi network -LRB- TGN -RRB- to the lysosome . Both GGA and AP proteins are directly recruited to the Golgi apparatus by GTP-bound Arf1 -LSB- 14 -RSB- . However , genes encoding GGA proteins and AP-2 are absent from the T. brucei genome -LSB- 2,15,16 -RSB- , implying either a greater dependence on the remaining AP-1 and AP-3 complexes or the operation of additional uncharacterized sorting mechanisms . Much of our current knowledge of lysosomal targeting in T. brucei stems from studies on p67 , a protein of unknown function that shares structural similarity but not sequence identity with the mammalian LAMP proteins -LSB- 17 -RSB- . This protein is trafficked from the ER via the Golgi to the lysosome , utilizing distinct targeting signals in different stages of the parasite life cycle . In PCF cells , the C-terminal cytoplasmic domain of p67 is necessary and sufficient for lysosomal targeting , whereas this region is not required in BSF cells -LSB- 8 -RSB- . While the cytoplasmic domain contains two putative dileucine motifs -LSB- 8 -RSB- , a recent study has analyzed these regions functionally in PCF and demonstrated that both are required to support maximal targeting -LSB- 18 -RSB- . It is likely that p67 is sorted in PCF cells via binding to either AP-1 or AP-3 at the TGN , a process that could be inhibited by knockdown of ARF1 . While the data presented here suggest that p67 continues to be targeted efficiently for degradation in the lysosome , this does not preclude a defect in the sorting or trafficking of other essential lysosomal factors . In BSF parasites , the AP complexes may not be directly involved in p67 sorting , given the redundancy of the dileucine motifs in this life cycle stage -LSB- 8 -RSB- . As in PCF cells , knockdown of TbARF1 does not prevent p67 localization to the lysosome -LSB- 2 -RSB- . However , in contrast , the inducible expression of a constitutively active form of TbARF1 does not inhibit endocytosis but prevents p67 from reaching the lysosome , resulting in extremely rapid cell death -LSB- 2 -RSB- . In this situation , excess ARF1 may be sequestering effector proteins required for alternative pathways which would normally facilitate the correct localization of p67 . Given that forward biosynthetic trafficking to the lysosome is not quantitatively impaired in the absence of ARF1 , this protein may alternatively influence the recycling of membranes from the lysosome , a defect that might generate multivesicular-type structures such as those observed at higher resolution in Fig. 2 . Further studies are required to pinpoint the relationship between TbARF1 and lysosomal function , analysis which may reveal insights into the secretory pathways of lower eukaryotes .

O O O O O O B O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

trypanosoma brucei
arf1
endocytosis
lysosome

arf, adp ribosylation factor
bsf, bloodstream form
pcf, procyclic form
rnai, rna interference
t. brucei, trypanosoma brucei