BACKGROUND
autophagy is an evolutionarily conserved intracellular process by which cytoplasmic constituents, including long-lived proteins, protein aggregates, organelles, and invading pathogens, are delivered to lysosomes for degradation and subsequent recycling  <cit> .
autophagy is activated in response to changes in the internal status of the cell and/or changes in the extracellular environment and plays an essential role in cell and tissue homeostasis.
deregulation of autophagy has been linked to many human diseases such as cancer, neurodegeneration, myopathies, diabetes, and infections by bacteria and viruses  <cit> .
genetic screens, primarily in the yeast s. cerevisiae, have identified numerous autophagy-related genes , many of which have orthologs in higher eukaryotes, including c. elegans, drosophila, and mammals  <cit> .
the products of these genes constitute two unique protein conjugation systems responsible for formation and elongation of autophagic isolation membranes during autophagy  <cit> .
in addition to the conjugation systems, autophagy initiation is also dependent on phosphatidylinositol 3-phosphate  based on the crucial role of the type iii pi3kinase vps <dig>  an enzyme that generates pi3p and forms a complex with autophagy regulator beclin- <dig>   <cit> .
the level of pi3p is also controlled by pi3p phosphatases that belong to the myotubularin family in mammalian cells  <cit> , which presumably play a role in regulation of autophagy as well.
indeed, studies have demonstrated that pi3p phosphatase jumpy and myotubularin-related phosphatase  <dig>  act as negative regulators of autophagy in mammalian cells  <cit> .
myotubularin phosphatases are members of the protein tyrosine phosphatase superfamily  <cit> .
but unlike other protein phosphatases, myotubularin family enzymes dephosphorylate pi3p and phosphatidylinositol -bi-phosphate  <cit> .
mutations in genes encoding myotubularin proteins are associated with diseases.
for example, mutations in mtm <dig>  the founding member of this family, cause x-linked myotubular myopathy , a severe congenital muscular disorder, while mutations in mtmr <dig> and mtmr <dig> are associated with charcot-marie-tooth disease  <cit> .
we previously have isolated and characterized a c. elegans homolog of myotubularin proteins, designated cemtm <dig>  cemtm <dig> preferably dephosphorylates pi3p and contains a fyve lipid-binding domain at its c-terminus which binds to pi3p  <cit> .
knockdown of cemtm <dig> in c. elegans worms by using feeding-based rna interference caused severe impairment of body movement following post-reproductive age and also significantly shortened their lifespan  <cit> .
we reasoned that this may be related to loss of muscle function due to de-regulation of autophagy.
in this study, we demonstrate that knockdown of cemtm <dig> induces autophagy that precedes an accelerated loss of muscle fibers in c. elegans worms.
RESULTS
knockdown of cemtm <dig> causes loss of muscle fibers in adult c. elegans
in an earlier study, we isolated and characterized a c. elegans homolog of myotubularin phosphatases, designated cemtm <dig>  <cit> .
cemtm <dig> is predominately expressed in muscle of adult c. elegans worms.
it binds pi3p through its c-terminal fyve domain and preferably dephosphorylates pi3p.
knockdown of cemtm <dig> by using feeding-based rna interference leads to near total loss of cemtm <dig> expression and causes a gradual impairment of body movement from day  <dig> with significant shortening of lifespan of the worms  <cit> .
since cemtm <dig> is predominantly expressed in the muscle  <cit> , the progressive locomotory impairment associated with knockdown of the enzyme may be caused by declining muscle functions.
to verify this, we employed alexa fluor 568-conjugated phalloidin to detect actin fibers in whole-mount worms .
on day  <dig>  both control and rnai-treated worms displayed clear and organized actin fibers.
however, on day  <dig>  clear deterioration of the fibers was seen with the rnai-treated worms, and by day  <dig> the fibers were essentially absent, which correlated with the total impairment of body movement.
in contrast, the control worms still maintained actin fiber structure on day  <dig>  although not as organized as that seen with younger worms.
by day  <dig>  control worms also displayed significant loss of muscle fibers.
loss of muscle fibers is a progressive event as the worm ages, but knockdown of cemtm <dig> markedly accelerates the process.
therefore, our data indicate that cemtm <dig> is required to stabilize muscle fibers in adult c. elegans worms.
to further verify the effects of cemtm <dig> knockdown on muscle fibers, we employed rw <dig> worms which express gfp:: mhc a carried by a construct in which a gfp coding sequence was inserted at the translation initiation codon in the gene for myosin heavy chain a  <cit> .
the data are shown in figure 1b.
on day  <dig>  both control and rnai-treated young adult worms showed strong and organized muscle fibers.
however, on day  <dig>  while the muscle fibers in control worms displayed a slight decrease, those in the cemtm rnai-treated worms were markedly reduced.
quantification of gfp fluorescence signals revealed near 50% loss of gfp-myosin in the treated worms on day  <dig>  the data provide further evidence that knockdown of cemtm <dig> destabilize muscle fibers which contain both myosin and actin.
knockdown of cemtm <dig> shortens the body size of adult c. elegans
besides the impairment of locomotion, knockdown of cemtm <dig> also affected the body size of c. elegans worms.
as shown in figure 2a and b, control c. elegans entered reproductive period on day  <dig>  but they continued to grow until around day  <dig> to day  <dig>  however, cemtm <dig> rnai-treated worms started to shrink after day  <dig>  on day  <dig>  the average size of cemtm <dig> rnai-treated worms were 8-15% shorter than the control worms of the same age.
the shortening of body length is apparently due to a reduction in the size of individual muscle cells as revealed by staining of muscle actin fibers with alexa fluor 568-conjugated phalloidin .
on average, the muscle cell size was reduced by about 30% .
it should be noted that knockdown of cemtm <dig> had no significant effects on the development of worms from embryos to young adults .
knockdown of cemtm <dig> causes occurrence of autophagy in multiple cells of adult c. elegans worms
we hypothesized that the loss of muscle fibers may be caused by death of muscle cells due to autophagy, which is known to have an important role in muscle maintenance and to be dependent on pi3p  <cit> .
our earlier studies have shown the knockdown of cemtm <dig> affects the total level of pi3p in c. elegans worms  <cit> .
it is likely that loss of cemtm function causes autophagy, thereby leading to loss of muscle fibers.
to visualize autophagy, we used worms carrying a gfp::lgg- <dig> fusion protein.
lgg- <dig> is an ortholog of yeast atg8p and mammalian map-lc <dig> in c. elegans.
during autophagy, gfp-labeled lgg- <dig> protein localizes to preautophagosomal and autophagosomal membranes and forms green puncta, thereby serving as a reporter for autophagy  <cit> .
data shown in figure  <dig> demonstrate occurrence of autophagy in multiple cells in cemtm <dig> rnai-treated worms on different days.
autophagy occurred in seam cells in day  <dig> larvae, in muscle cells in day  <dig> young adults, and day  <dig> and thereafter in intestine of adult worms .
in the control worm, such structures were essentially absent until day  <dig> when a few sporadic punctuates appeared in the intestine .
we also confirmed induction of autophagy by detecting lipidation of gfp-lgg- <dig>  another hallmark of autophagy.
as shown in figure 3e, upon treatment of c. elegans worms with cemtm <dig> rnai, a clear band of ~ <dig> kda was recognized by anti-gfp antibody in the membrane extracts of worms.
together, our data indicate that knockdown of cemtm <dig> caused a marked increase in autophagy activity.
it is worth noting that the onset of autophagy apparently preceded the deterioration of muscle fibers which began on day  <dig> .
apparently, the presence of cemtm <dig> prevents occurrence of autophagy in the normal worms as they age.
the occurrence of autophagy in muscle and intestine is consistent with the distributions of the enzyme in these tissues as revealed in our earlier studies  <cit> .
knockdown cemtm <dig> increases lysosomal activities and induces necrotic cell death in c. elegans
after formation, autophagosomes deliver the sequestered cytoplasmic materials into lysosomes for further degradation  <cit> .
to find out whether the formation of autophagosomes induced by knockdown of cemtm <dig> has further functional consequences, we examined the lysosomal activities by using acridine orange, which is commonly used for staining of lysosomes  <cit> .
the results showed that knockdown of cemtm <dig> induced appearance of numerous lysosomal vesicles in the head region of day  <dig> adult worms .
these vesicles were apparently in muscle cells as the muscle fibers were visible in dic images.
the lysosomal vesicles were also seen in the body wall muscle although less predominant .
excessive lysosomes induce cytoplasmic acidification and further lead to necrotic cell death.
in fact, studies have demonstrated that autophagy is required for necrotic cell death in c. elegans <cit> .
as expected, knockdown of cemtm <dig> caused significant necrosis as shown in figure  <dig>  in the head region of cemtm <dig> rnai-treated worms, necrotic cell death, as indicated by cell corpses, started to appear on day  <dig> and became overwhelming by day  <dig>  however, this was not seen in control worms, although sporadic cell necrotic cell death was also occasionally observed on day  <dig> and thereafter.
on average, control day  <dig> worms displayed  <dig> ± <dig>  corpses in the head region while cemtm <dig> rnai-treated worms of the same age showed 4± <dig>  corpses .
necrotic cell death was also seen in the body wall muscle of rnai-treated worms on day  <dig> .
knockdown cemtm <dig> does not aggravate the abnormalities of muscle wasting in autophagy-deficient worms
the above data suggest knockdown of cemtm <dig> function leads to autophagy which in turn causes cell death and loss of muscle fibers.
to further address the causal relationship between loss of muscle fibers and onset of excessive autophagy, we employed autophagy deficient vc <dig> worms.
these worms lack atp- <dig>  an ortholog of yeast atg18p and human wipi, and thus are not able to recruit autophagic membranes to form autophagosomes  <cit> .
interestingly, vc <dig> worms fed on normal e. coli food exhibited impaired locomotion after day  <dig> .
this was associated with deterioration of muscle fibers as revealed by alexa fluor 568-conjugated phalloidin staining .
nonetheless, knockdown of cemtm <dig> did not further exacerbate this abnormality, implying that induction of autophagy is required for cemtm <dig> rnai-induced muscle fiber loss.
the data described above demonstrated that knockdown of a mtm family enzyme causes autophagy and subsequently causes loss of muscle fibers.
as a pi3p phosphatase, cemtm <dig> should play an important role in controlling the level of pi3p.
the involvement of pi3p in autophagy has been demonstrated for the most part by studying the type iii pi3kinase vps <dig>  vps <dig> is a type iii pi <dig> kinase responsible for generation of pi3p.
it forms a complex with its autophagy-regulatory partner beclin- <dig> , thereby initiating autophagy  <cit> .
vps <dig> also has an essential role in membrane trafficking and endocytosis, and vps <dig> mutant worms display lethality and molting defects as well as alterations in the outer nuclear membrane and in the endoplasmic reticulum  <cit> .
our current study demonstrated that knockdown of cemtm <dig>  a pi3p phosphatase predominately expressed in the muscle, results in autophagy which subsequently leads to loss of muscle fibers.
in a sense, one may postulate that inactivation of cemtm <dig> is equivalent to activation of vps <dig> in regulating the autophagy process.
therefore, pi3p phosphatases and pi <dig> kinases play equally important roles in regulation of autophagy.
the role of autophagy in muscle wasting and maintenance has been well documented  <cit> .
autophagy appears to be a double edged sword.
too much or too little of it can cause muscle weakness and atrophy.
for example, excessive autophagy, induced by denervation and fasting, contributes to muscle atrophy  <cit> .
by demonstrating that occurrence of autophagy upon knockdown of cemtm <dig> precedes muscle fiber loss, our study provides further proof of the harmful effects of too much autophagy in muscle function.
on the other hand, deficiency in autophagy is also known to cause muscle atrophy.
for example, both atg5−/− and atg7−/− mice show muscle loss accompanied by accumulation of protein aggregates and abnormal membranous structures in the muscle cells  <cit> .
by the same concept, our study demonstrated that autophagy-deficient atg- <dig> worms exhibited impaired body movement and loss of muscle fibers .
in addition, an earlier study demonstrated that loss of autophagy function due to inactivation of unc-51/atg <dig> and bec-1/atg6/beclin <dig> results in small body size without affecting cell number  <cit> .
our current data demonstrated that excessive autophagy associated with knockdown of cemtm <dig> also causes shortening of body size with smaller muscle cells.
therefore, autophagy appears to be a dynamic process and should be kept in a balance.
from a therapeutic perspective, it is important to understand whether the reduction of autophagy is helpful during muscle loss or whether enhancement of autophagosome flux is beneficial for the clearance of dangerous organelles or toxic proteins.
to keep healthy muscle function, we need to keep a normal level of autophagy flux to rejuvenate organelles and to remove dysfunctional mitochondria and er membranes but to avoid excessive autophagy which may lead to breakdown of normal muscle fibers.
the loss of muscle mass, referred to as sarcopenia, is a normal phenomenon in animals as a consequence of aging  <cit> .
for humans, decrease in muscle tissue begins around the age of  <dig> years and becomes more dramatic beyond the 60th year of life.
for c. elegans worms, loss of muscle fibers and consequent impairment of body movement is clearly seen after day  <dig> and is much accelerated by knockdown of cemtm <dig> .
aging is associated with a progressive decline of muscle mass, strength, and quality, but the mechanism underlying the muscle wasting remains unresolved  <cit> .
studies have shown that sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle  <cit> , implying that the maintenance of existing muscle is crucial.
among the many factors correlating with sarcopenia during aging, oxidative stress has been extensively investigated  <cit> .
we believe that oxidation stress-induced inactivation of mtm family phosphatases may play a major role in the muscle wasting process.
like all the other members of the tyrosine phosphatase superfamily, mtm family enzymes are susceptible to oxidation-induced inactivation because they contain a highly reactive cysteinyl residue at the catalytic center.
reactive oxygen species, such as the superoxide radical , hydrogen peroxide , the hydroxyl radical , and nitric oxide  are produced in muscle at rest, and this generation is increased by contractile activity  <cit> .
oxidative damage is considered the main cause of aging.
in fact, it was thought that a slower accumulation of oxidative damage is at least partly responsible for the life extension effects seen in c. elegans with daf- <dig> and age- <dig> mutations  <cit> .
our earlier data have shown that cemtm <dig> is sensitive to peroxide and loss of cemtm <dig> function causes muscle deterioration in c. elegans <cit> .
therefore, inactivation of cemtm <dig> by oxidation may be attributable to loss of muscle fibers in normal worms at later ages.
thus, to prevent sarcopenia, it is important to maintain a normal level of pi3p phosphatase activity.
CONCLUSIONS
by using the c. elegans worm as a model system, our study provides further evidence that the mtm family phosphatases play a negative role in autophagy.
our study also demonstrates a correlation between excessive autophagy and muscle wasting, implying a normal level of autophagy is important for muscle function and maintenance.
considering sensitivity of tyrosine phosphatase superfamily enzymes to reactive oxygen species, loss of muscle fibers during normal aging may be a consequence of pi3p phosphatase inactivation.
therefore, studying the mtm enzymes may have clinical implications in the prevention and treatment of human sarcopenia.

 autophagy is an evolutionarily conserved intracellular process by which cytoplasmic constituents, including long-lived proteins, protein aggregates, organelles, and invading pathogens, are delivered to lysosomes for degradation and subsequent recycling. autophagy is activated in response to changes in the internal status of the cell and/or changes in the extracellular environment and plays an essential role in cell and tissue homeostasis. deregulation of autophagy has been linked to many human diseases such as cancer, neurodegeneration, myopathies, diabetes, and infections by bacteria and viruses. genetic screens, primarily in the yeast s. cerevisiae, have identified numerous autophagy-related genes, many of which have orthologs in higher eukaryotes, including c. elegans, drosophila, and mammals. the products of these genes constitute two unique protein conjugation systems responsible for formation and elongation of autophagic isolation membranes during autophagy. in addition to the conjugation systems, autophagy initiation is also dependent on phosphatidylinositol 3-phosphate based on the crucial role of the type iii pi3kinase vps an enzyme that generates pi3p and forms a complex with autophagy regulator beclin-. the level of pi3p is also controlled by pi3p phosphatases that belong to the myotubularin family in mammalian cells, which presumably play a role in regulation of autophagy as well. indeed, studies have demonstrated that pi3p phosphatase jumpy and myotubularin-related phosphatase act as negative regulators of autophagy in mammalian cells. myotubularin phosphatases are members of the protein tyrosine phosphatase superfamily. but unlike other protein phosphatases, myotubularin family enzymes dephosphorylate pi3p and phosphatidylinositol -bi-phosphate. mutations in genes encoding myotubularin proteins are associated with diseases. for example, mutations in mtm the founding member of this family, cause x-linked myotubular myopathy, a severe congenital muscular disorder, while mutations in mtmr and mtmr are associated with charcot-marie-tooth disease. we previously have isolated and characterized a c. elegans homolog of myotubularin proteins, designated cemtm cemtm preferably dephosphorylates pi3p and contains a fyve lipid-binding domain at its c-terminus which binds to pi3p. knockdown of cemtm in c. elegans worms by using feeding-based rna interference caused severe impairment of body movement following post-reproductive age and also significantly shortened their lifespan. we reasoned that this may be related to loss of muscle function due to de-regulation of autophagy. in this study, we demonstrate that knockdown of cemtm induces autophagy that precedes an accelerated loss of muscle fibers in c. elegans worms.  knockdown of cemtm causes loss of muscle fibers in adult c. elegans in an earlier study, we isolated and characterized a c. elegans homolog of myotubularin phosphatases, designated cemtm. cemtm is predominately expressed in muscle of adult c. elegans worms. it binds pi3p through its c-terminal fyve domain and preferably dephosphorylates pi3p. knockdown of cemtm by using feeding-based rna interference leads to near total loss of cemtm expression and causes a gradual impairment of body movement from day with significant shortening of lifespan of the worms. since cemtm is predominantly expressed in the muscle, the progressive locomotory impairment associated with knockdown of the enzyme may be caused by declining muscle functions. to verify this, we employed alexa fluor 568-conjugated phalloidin to detect actin fibers in whole-mount worms. on day both control and rnai-treated worms displayed clear and organized actin fibers. however, on day clear deterioration of the fibers was seen with the rnai-treated worms, and by day the fibers were essentially absent, which correlated with the total impairment of body movement. in contrast, the control worms still maintained actin fiber structure on day although not as organized as that seen with younger worms. by day control worms also displayed significant loss of muscle fibers. loss of muscle fibers is a progressive event as the worm ages, but knockdown of cemtm markedly accelerates the process. therefore, our data indicate that cemtm is required to stabilize muscle fibers in adult c. elegans worms. to further verify the effects of cemtm knockdown on muscle fibers, we employed rw worms which express gfp:: mhc a carried by a construct in which a gfp coding sequence was inserted at the translation initiation codon in the gene for myosin heavy chain a. the data are shown in figure 1b. on day both control and rnai-treated young adult worms showed strong and organized muscle fibers. however, on day while the muscle fibers in control worms displayed a slight decrease, those in the cemtm rnai-treated worms were markedly reduced. quantification of gfp fluorescence signals revealed near 50% loss of gfp-myosin in the treated worms on day the data provide further evidence that knockdown of cemtm destabilize muscle fibers which contain both myosin and actin. knockdown of cemtm shortens the body size of adult c. elegans besides the impairment of locomotion, knockdown of cemtm also affected the body size of c. elegans worms. as shown in figure 2a and b, control c. elegans entered reproductive period on day but they continued to grow until around day to day however, cemtm rnai-treated worms started to shrink after day on day the average size of cemtm rnai-treated worms were 8-15% shorter than the control worms of the same age. the shortening of body length is apparently due to a reduction in the size of individual muscle cells as revealed by staining of muscle actin fibers with alexa fluor 568-conjugated phalloidin. on average, the muscle cell size was reduced by about 30%. it should be noted that knockdown of cemtm had no significant effects on the development of worms from embryos to young adults. knockdown of cemtm causes occurrence of autophagy in multiple cells of adult c. elegans worms we hypothesized that the loss of muscle fibers may be caused by death of muscle cells due to autophagy, which is known to have an important role in muscle maintenance and to be dependent on pi3p. our earlier studies have shown the knockdown of cemtm affects the total level of pi3p in c. elegans worms. it is likely that loss of cemtm function causes autophagy, thereby leading to loss of muscle fibers. to visualize autophagy, we used worms carrying a gfp::lgg- fusion protein. lgg- is an ortholog of yeast atg8p and mammalian map-lc in c. elegans. during autophagy, gfp-labeled lgg- protein localizes to preautophagosomal and autophagosomal membranes and forms green puncta, thereby serving as a reporter for autophagy. data shown in figure demonstrate occurrence of autophagy in multiple cells in cemtm rnai-treated worms on different days. autophagy occurred in seam cells in day larvae, in muscle cells in day young adults, and day and thereafter in intestine of adult worms. in the control worm, such structures were essentially absent until day when a few sporadic punctuates appeared in the intestine. we also confirmed induction of autophagy by detecting lipidation of gfp-lgg- another hallmark of autophagy. as shown in figure 3e, upon treatment of c. elegans worms with cemtm rnai, a clear band of ~ kda was recognized by anti-gfp antibody in the membrane extracts of worms. together, our data indicate that knockdown of cemtm caused a marked increase in autophagy activity. it is worth noting that the onset of autophagy apparently preceded the deterioration of muscle fibers which began on day. apparently, the presence of cemtm prevents occurrence of autophagy in the normal worms as they age. the occurrence of autophagy in muscle and intestine is consistent with the distributions of the enzyme in these tissues as revealed in our earlier studies. knockdown cemtm increases lysosomal activities and induces necrotic cell death in c. elegans after formation, autophagosomes deliver the sequestered cytoplasmic materials into lysosomes for further degradation. to find out whether the formation of autophagosomes induced by knockdown of cemtm has further functional consequences, we examined the lysosomal activities by using acridine orange, which is commonly used for staining of lysosomes. the results showed that knockdown of cemtm induced appearance of numerous lysosomal vesicles in the head region of day adult worms. these vesicles were apparently in muscle cells as the muscle fibers were visible in dic images. the lysosomal vesicles were also seen in the body wall muscle although less predominant. excessive lysosomes induce cytoplasmic acidification and further lead to necrotic cell death. in fact, studies have demonstrated that autophagy is required for necrotic cell death in c. elegans. as expected, knockdown of cemtm caused significant necrosis as shown in figure in the head region of cemtm rnai-treated worms, necrotic cell death, as indicated by cell corpses, started to appear on day and became overwhelming by day however, this was not seen in control worms, although sporadic cell necrotic cell death was also occasionally observed on day and thereafter. on average, control day worms displayed ± corpses in the head region while cemtm rnai-treated worms of the same age showed 4± corpses. necrotic cell death was also seen in the body wall muscle of rnai-treated worms on day. knockdown cemtm does not aggravate the abnormalities of muscle wasting in autophagy-deficient worms the above data suggest knockdown of cemtm function leads to autophagy which in turn causes cell death and loss of muscle fibers. to further address the causal relationship between loss of muscle fibers and onset of excessive autophagy, we employed autophagy deficient vc worms. these worms lack atp- an ortholog of yeast atg18p and human wipi, and thus are not able to recruit autophagic membranes to form autophagosomes. interestingly, vc worms fed on normal e. coli food exhibited impaired locomotion after day. this was associated with deterioration of muscle fibers as revealed by alexa fluor 568-conjugated phalloidin staining. nonetheless, knockdown of cemtm did not further exacerbate this abnormality, implying that induction of autophagy is required for cemtm rnai-induced muscle fiber loss. the data described above demonstrated that knockdown of a mtm family enzyme causes autophagy and subsequently causes loss of muscle fibers. as a pi3p phosphatase, cemtm should play an important role in controlling the level of pi3p. the involvement of pi3p in autophagy has been demonstrated for the most part by studying the type iii pi3kinase vps vps is a type iii pi kinase responsible for generation of pi3p. it forms a complex with its autophagy-regulatory partner beclin-, thereby initiating autophagy. vps also has an essential role in membrane trafficking and endocytosis, and vps mutant worms display lethality and molting defects as well as alterations in the outer nuclear membrane and in the endoplasmic reticulum. our current study demonstrated that knockdown of cemtm a pi3p phosphatase predominately expressed in the muscle, results in autophagy which subsequently leads to loss of muscle fibers. in a sense, one may postulate that inactivation of cemtm is equivalent to activation of vps in regulating the autophagy process. therefore, pi3p phosphatases and pi kinases play equally important roles in regulation of autophagy. the role of autophagy in muscle wasting and maintenance has been well documented. autophagy appears to be a double edged sword. too much or too little of it can cause muscle weakness and atrophy. for example, excessive autophagy, induced by denervation and fasting, contributes to muscle atrophy. by demonstrating that occurrence of autophagy upon knockdown of cemtm precedes muscle fiber loss, our study provides further proof of the harmful effects of too much autophagy in muscle function. on the other hand, deficiency in autophagy is also known to cause muscle atrophy. for example, both atg5−/− and atg7−/− mice show muscle loss accompanied by accumulation of protein aggregates and abnormal membranous structures in the muscle cells. by the same concept, our study demonstrated that autophagy-deficient atg- worms exhibited impaired body movement and loss of muscle fibers. in addition, an earlier study demonstrated that loss of autophagy function due to inactivation of unc-51/atg and bec-1/atg6/beclin results in small body size without affecting cell number. our current data demonstrated that excessive autophagy associated with knockdown of cemtm also causes shortening of body size with smaller muscle cells. therefore, autophagy appears to be a dynamic process and should be kept in a balance. from a therapeutic perspective, it is important to understand whether the reduction of autophagy is helpful during muscle loss or whether enhancement of autophagosome flux is beneficial for the clearance of dangerous organelles or toxic proteins. to keep healthy muscle function, we need to keep a normal level of autophagy flux to rejuvenate organelles and to remove dysfunctional mitochondria and er membranes but to avoid excessive autophagy which may lead to breakdown of normal muscle fibers. the loss of muscle mass, referred to as sarcopenia, is a normal phenomenon in animals as a consequence of aging. for humans, decrease in muscle tissue begins around the age of years and becomes more dramatic beyond the 60th year of life. for c. elegans worms, loss of muscle fibers and consequent impairment of body movement is clearly seen after day and is much accelerated by knockdown of cemtm. aging is associated with a progressive decline of muscle mass, strength, and quality, but the mechanism underlying the muscle wasting remains unresolved. studies have shown that sarcopenia is not due to lack of regenerative drive in senescent skeletal muscle, implying that the maintenance of existing muscle is crucial. among the many factors correlating with sarcopenia during aging, oxidative stress has been extensively investigated. we believe that oxidation stress-induced inactivation of mtm family phosphatases may play a major role in the muscle wasting process. like all the other members of the tyrosine phosphatase superfamily, mtm family enzymes are susceptible to oxidation-induced inactivation because they contain a highly reactive cysteinyl residue at the catalytic center. reactive oxygen species, such as the superoxide radical, hydrogen peroxide, the hydroxyl radical, and nitric oxide are produced in muscle at rest, and this generation is increased by contractile activity. oxidative damage is considered the main cause of aging. in fact, it was thought that a slower accumulation of oxidative damage is at least partly responsible for the life extension effects seen in c. elegans with daf- and age- mutations. our earlier data have shown that cemtm is sensitive to peroxide and loss of cemtm function causes muscle deterioration in c. elegans. therefore, inactivation of cemtm by oxidation may be attributable to loss of muscle fibers in normal worms at later ages. thus, to prevent sarcopenia, it is important to maintain a normal level of pi3p phosphatase activity.  by using the c. elegans worm as a model system, our study provides further evidence that the mtm family phosphatases play a negative role in autophagy. our study also demonstrates a correlation between excessive autophagy and muscle wasting, implying a normal level of autophagy is important for muscle function and maintenance. considering sensitivity of tyrosine phosphatase superfamily enzymes to reactive oxygen species, loss of muscle fibers during normal aging may be a consequence of pi3p phosphatase inactivation. therefore, studying the mtm enzymes may have clinical implications in the prevention and treatment of human sarcopenia. 
our data suggest an important role of cemtm in regulating autophagy and maintaining muscle fibers. this was preceded by the occurrence of excessive autophagy in the muscle and other tissues, which subsequently resulted in increased lysosomal activity and necrotic cell death. knockdown of cemtm by using feeding-based rna interference caused loss of muscle fibers accompanied by shortening of muscle cell and body size in aged c. elegans worms. in this study, we demonstrate that cemtm a fyve-domain containing myotubalarin family phosphatase, is required for the maintenance of muscle fibers by preventing excessive autophagy in caenorhabditis elegans. this study may have clinical implications for prevention and treatment of sarcopenia.
 autophagy is a ubiquitous cellular process responsible for the bulk degradation of cytoplasmic components through the autophagosomal-lysosomal pathway. in skeletal muscle, autophagy has been regarded as a key regulator for muscle mass maintenance, and its imbalance leads to sarcopenia. however, the underlying mechanism is poorly understood.  in this study, we demonstrate that cemtm a fyve-domain containing myotubalarin family phosphatase, is required for the maintenance of muscle fibers by preventing excessive autophagy in caenorhabditis elegans. knockdown of cemtm by using feeding-based rna interference caused loss of muscle fibers accompanied by shortening of muscle cell and body size in aged c. elegans worms. this was preceded by the occurrence of excessive autophagy in the muscle and other tissues, which subsequently resulted in increased lysosomal activity and necrotic cell death. however, knockdown of cemtm did not aggravate the abnormalities of muscle wasting in autophagy-deficient atg- mutant worms.  our data suggest an important role of cemtm in regulating autophagy and maintaining muscle fibers. this study may have clinical implications for prevention and treatment of sarcopenia. phosphatasemyotubalarinrnaiautophagymusclec. eleganssarcopenia 