The Deubiquitinating Enzyme AMSH1 and the ESCRT-III Subunit VPS2.1 Are Required for Autophagic Degradation in Arabidopsis

In eukaryotes, posttranslational modification by ubiquitin regulates the activity and stability of many proteins and thus influences a variety of developmental processes as well as environmental responses. Ubiquitination also plays a critical role in intracellular trafficking by serving as a signal for endocytosis. We have previously shown that the Arabidopsis thaliana ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM3 (AMSH3) is a deubiquitinating enzyme (DUB) that interacts with ENDOSOMAL COMPLEX REQUIRED FOR TRANSPORT-III (ESCRT-III) and is essential for intracellular transport and vacuole biogenesis. However, physiological functions of AMSH3 in the context of its ESCRT-III interaction are not well understood due to the severe seedling lethal phenotype of its null mutant. In this article, we show that Arabidopsis AMSH1, an AMSH3-related DUB, interacts with the ESCRT-III subunit VACUOLAR PROTEIN SORTING2.1 (VPS2.1) and that impairment of both AMSH1 and VPS2.1 causes early senescence and hypersensitivity to artificial carbon starvation in the dark similar to previously reported autophagy mutants. Consistent with this, both mutants accumulate autophagosome markers and accumulate less autophagic bodies in the vacuole. Taken together, our results demonstrate that AMSH1 and the ESCRT-III-subunit VPS2.1 are important for autophagic degradation and autophagy-mediated physiological processes.     

The Deubiquitinating Enzyme AMSH3 Is Required for Intracellular Trafficking and Vacuole Biogenesis in Arabidopsis thaliana

Ubiquitination, deubiquitination, and the formation of specific ubiquitin chain topologies have been implicated in various cellular processes. Little is known, however, about the role of ubiquitin in the development of cellular organelles. Here, we identify and characterize the deubiquitinating enzyme AMSH3 from Arabidopsis thaliana. AMSH3 hydrolyzes K48- and K63-linked ubiquitin chains in vitro and accumulates both ubiquitin chain types in vivo. amsh3 mutants fail to form a central lytic vacuole, accumulate autophagosomes, and mis-sort vacuolar protein cargo to the intercellular space. Furthermore, AMSH3 is required for efficient endocytosis of the styryl dye FM4-64 and the auxin efflux facilitator PIN2. We thus present evidence for a role of deubiquitination in intracellular trafficking and vacuole biogenesis.     

Response to novel food and the role of social influences in common marmosets (Callithrix jacchus) and Goeldi's monkeys (Callimico goeldii)

Neophobia, defined as showing caution toward novel features of the environment, is widespread in birds and mammals; it can be affected by ecology, early experience, and social context. In this study, we aimed to (i) investigate the response to novel food in adult common marmosets and Goeldi's monkeys and (ii) assess the role of social influences. We used an experimental paradigm employed previously with capuchin monkeys and children, in which a subject (observer) was presented with a novel food under three conditions: (i) Presence: group members did not have food; (ii) Different color: group members received familiar food whose color differed from that of the observer's novel food; (iii) Same color: group members received familiar food of the same color as the observer's novel food. Although most common marmosets tasted and/or ate the novel food, none of the Goeldi's monkeys ate it and only two sampled it. Differences in home range size and early social experience might explain the divergent behavior of the two species. Observers of both species similarly attended to group members and their visual attention increased with the number of group members eating, especially when the observer's and group members' foods were perceptually similar. However, we observed social influences on explorative behavior in Goeldi's monkeys but not on explorative or eating behavior in common marmosets. This result might be explained by the different pattern of response to novel food observed in the two species. Moreover, social influences on Goeldi's monkeys' behavior were nonspecific, i.e. they were not based on an appreciation that the food is safe because eaten by group members.     

The Arabidopsis deubiquitinating enzyme AMSH3 interacts with ESCRT-III subunits and regulates their localization

Ubiquitination and deubiquitination regulate various cellular processes. We have recently shown that the deubiquitinating enzyme Associated Molecule with the SH3 domain of STAM3 (AMSH3) is involved in vacuole biogenesis and intracellular trafficking in Arabidopsis thaliana. However, little is known about the identity of its interaction partners and deubiquitination substrates. Here, we provide evidence that AMSH3 interacts with ESCRT-III subunits VPS2.1 and VPS24.1. The interaction of ESCRT-III subunits with AMSH3 is mediated by the MIM1 domain and depends on the MIT domain of AMSH3. We further show that AMSH3, VPS2.1, and VPS24.1 localize to class E compartments when ESCRT-III disassembly is inhibited by coexpression of inactive Suppressor of K+ transport Defect 1 (SKD1), an AAA-ATPase involved in the disassembly of ESCRT-III. We also provide evidence that AMSH3 and SKD1 compete for binding to VPS2.1. Furthermore, we show that the loss of AMSH3 enzymatic activity leads to the formation of cellular compartments that contain AMSH3, VPS2.1, and VPS24.1. Taken together, our study presents evidence that AMSH3 interacts with classical core ESCRT-III components and thereby provides a molecular framework for the function of AMSH3 in plants.     

Kinematic Analysis of Trunk-to-trunk Leaping in <Emphasis Type="Italic">Callimico goeldii</Emphasis>

Leaping to and from vertical trunks is a pattern of locomotor behavior that characterizes the positional repertoire of several prosimian and neotropical primate species. We examined the kinematics of leaping in a group of 6 captive Goeldis monkeys. We introduced a set of 2 wooden, fixed, non-compliant vertical supports in their enclosure and used 2 video cameras set at right angles to document leaping. The supports are 2.5, 6, or 15 cm in diameter and were placed at distances of between 1 and 2 m. We conducted frame-by-frame analyses of 122 leaps. The results indicate that irrespective of distance leaped and the diameter of takeoff and landing substrates, the forelimbs of {Callimico} contacted the landing platform in advance of the hind limbs. Moreover, even when leaping a horizontal distance of 2 m, {Callimico} experienced a downward vertical displacement of only 0.17 m. Several features of the shoulder and forelimb of {Callimico} appear to be associated with enhanced stability at the humeral head and radioulnar joint, and are consistent with the ability to withstand large compressive forces generated when landing on noncompliant substrates. Based on a series of kinematic equations provided by Warren and Crompton (1998a), the mechanical cost of transport in {Callimico} (5.4 m/s^–2) is greater than those of prosimian vertical clingers and leapers. However, compared to other callitrichine primates, {Callimico goeldii} is behaviorally and anatomically specialized for leaping between vertical trunks in the lowest layers of the forest understory.     

Development of prolactin levels in marmoset males: from adult son to first-time father

Previous studies have found a clear relationship between prolactin (prl) and paternal care in various vertebrate taxa. In New World monkeys, it has been demonstrated in several species that fathers have high prolactin levels even during periods without infant rearing. In this study, we followed the reproductive careers of common marmoset males as they transitioned from being an adult son within their native family to fathering their own offspring for the first time. Specifically, we examined the first experience of elevated prolactin levels in marmoset males. Additionally, we investigated the effects of the total number of experienced births as well as of age on prolactin levels. Our results show that common marmoset males did not experience an increase in prolactin secretion after pairing or shortly before birth of their first infants. However, prolactin levels rose more than twofold after the birth of their first infants and had lowered again 2.5 months after this event. We found no correlation between prolactin levels and the number of previous births experienced or age. Our study demonstrates that further work about a possible enhancing effect of prolactin on paternal care, by means of experimentally reducing hormonal levels, should be conducted in common marmosets using first-time fathers before males experience the first paternal increase in prolactin levels.     

Different ovarian responses to potential mates underlie species-specific breeding strategies in common marmoset and Goeldi's monkey

Callitrichids are cooperative breeders, characterized by obligate twinning, extensive paternal care, and monopolization of reproduction by the dominant female. This is the case in the common marmoset, and in common marmoset groups of more than one adult female, subordinate females are typically acyclic consistent with infertility. However, one callitrichid, Goeldi's monkey, gives birth to singletons and exhibits low paternal care. Given these reproductive traits of Goeldi's monkey, we hypothesized that there would not be suppression of ovarian activity. To test this hypothesis, we applied non-invasive endocrine methods in a step-wise experiment with laboratory groups of both species. In each species, six pairs of sisters were studied alone, in visual contact with an unrelated male and in a polygynous trio with the male, and urine samples were collected for determination of oestrogen titres reflecting ovarian activity. Common marmoset sister pairs exhibited a marked difference in social status: during the study 5 of 6 dominant females conceived but only 1 of 6 subordinate females; the remaining 5 subordinates were acyclic at the end of the study, and instances of ovulation typically resulted in aggression. Goeldi's monkey sister pairs showed no status differences: in all pairs, however, both sisters exhibited a temporary cessation of ovarian cyclicity on trio formation, followed by ovulation and conception. We conclude that these marked differences in ovarian responses reflect the differences in inter-female competition for paternal caregiving resources. In common marmosets with high inter-female competition, suppression of ovulation functions to reduce aggression received by subordinate females; in Goeldi's monkey with low competition, temporary cessation of ovulation could facilitate female choice.     

The pairbond in the titi monkey (Callicebus moloch): intrinsic versus extrinsic contributions of the pairmates

Aspects of a monogamous relationship can be inferred by observing reactions of mated individuals to unfamiliar conspecifics. The present study examined the behaviours shown by male and female titi monkeys (Callicebus moloch) toward an opposite-sex stranger, and the effect of their pairmate's absence or presence on these encounters. Behaviours within unfamiliar heterosexual pairs showed superficial similarities to behaviours of mated pairs. Although there were few affiliative behaviours between unfamiliar individuals, animals were often in spatial proximity and even performed sexual behaviour. The visual presence of a pairmate clearly affected encounters between unfamiliar heterosexual pairs in several respects. The presence of a male pairmate had a much stronger influence on all behavioural measures compared to the presence of a female pairmate. Possible functional aspects of these results are discussed.     

Identification of New World monkey MHC-DRB alleles using PCR,DGGE and direct sequencing

Identification of New World monkey MHC-DRB alleles has previously relied upon labor-intensive cloning and sequencing techniques. Here we describe a rapid and unambiguous way to distinguish DRB alleles in New World monkeys using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and direct sequencing. The highly variable second exon of New World monkey DRB alleles was amplified using generic DRB primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and alleles were determined using fluorescent-based sequencing. The validity of this typing procedure was confirmed by the identification of all DRB alleles previously characterized by cloning and sequencing techniques from an individual cotton-top tamarin. Importantly, our analysis revealed DRB alleles not previously identified in this reference animal. Following validation of our technique, the protocol was employed for the characterization of MHC-DRB alleles in four other species of New World monkey: the pygmy marmoset, white-faced saki monkey, long-haired spider monkey and owl monkey. Using this technique, we identified five alleles from the cotton-top tamarin, five alleles from the owl monkey, three alleles from the long-haired spider monkey, three alleles from the white-faced saki monkey and two alleles from the pygmy marmoset. On the basis of phylogenetic tree analyses, 13 new DRB alleles were assigned to eight different MHC-DRB lineages. Whereas traditional DRB typing via cloning and sequencing provides limited information, our new technique provides a simple and relatively rapid way of identifying New World monkey MHC-DRB alleles.Nucleotide sequence data reported are available in the GenBank/EMBL/DDBJ databases under the accession numbers AJ544165–AJ544177