Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

<p>Abstract</p> <p>Background</p> <p>Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library.</p> <p>Results</p> <p>Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1). Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids.</p> <p>Conclusion</p> <p>We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor. TLRR is homologous to a class of regulatory subunits for PP1, a central phosphatase in the reversible phosphorylation of proteins that is key to modulation of many intracellular processes. TLRR may serve to target this important signaling molecule near the nucleus of developing spermatids in order to control the cellular rearrangements of spermiogenesis.</p

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation

Functional adaptation of bone: The mechanostat and beyond

The conceptual model of the mechanostat proposed by Harold Frost in 1983 is among the most significant contributions to musculoskeletal research today. This model states that bone and other musculoskeletal tissues including cartilage, tendon and muscle respond to habitual exercise/loading and that changes in the loading environment lead to adequate structural adaptation of (bone) tissue architecture. The analogy with a thermostat clearly indicates presence of a physiological feedback system which is able to adjust bone mass and structure according to the engendered loads. In the bioengineering community, the mechanostat has been mathematically formulated as a feedback algorithm using a set point criterion based on a particular mechanical quantity such as strain, strain energy density among others. As pointed out by Lanyon and Skerry, while it is widely thought that in a single individual, there exists a single mechanostat set point, this view is flawed by the fact that different bones throughout the skeleton require a specific strain magnitude to maintain bone mass. Consequently, different bones respond differently to increases or decreases in loading depending on the sensitivity of the mechanostat. Osteocytes, i.e., cells embedded in the bone matrix are believed to be the major bone cells involved in sensing and transduction of mechanical loads. The purpose of this chapter is to review the concept of the mechanostat and its role in bone pathophysiology. To do this we provide examples of why and how the skeleton responds to complex loading stimuli made up of numerous different parameters including strain magnitude, frequency and rest intervals among others. We describe latest in vivo and ex vivo loading models, which allow exploration of various mechanobiological relations in the mechanostat model utilising controlled mechanical environments. A review of the bone cells and signalling transduction cascades involved in mechanosensation and bone adaptation will also be provided. Furthermore, we will discuss the mechanostat in a clinical context, e.g., how factors such as sex, age, genetic constitution, concomitant disease, nutrient availability, and exposure to drugs all affect bone’s response to mechanical loading. Understanding the mechanostat and mechanobiological regulatory factors involved in mechanosensation and desensitisation is essential for our ability to control bone mass based on physiological loading, either directly through different exercise regimens, or by manipulating bone cells in a targeted manner using tailored site and individual specific stimuli including pharmaceuticals