has been successfully applied in the isolation of tumoral cells,
neurons affected by Parkinson or Alzheimer diseases, virus-infected
cells, etc. [2–4]. Moreover, LCM was shown to be able to separate
cellular organelles from specific cells [5], growth cones [6], and
invadopodia [7]. One advantage of using LCM to enrich a sample
with the cells of interest is the possibility of performing subsequent
downstream studies of DNA, RNA, or protein composition.
Proteomics, the study of proteins, is one of the most important
technologies used to acquire new insights into cellular function
since proteins are the primary effectors of all biological activity.
However, the feasibility of combining proteomic techniques with
LCM depends primarily on the sensitivity of the technique used, as
the amount of protein retrieved from the dissected sample can be
very small. Traditional proteomic techniques cannot solve this
problem since they require a high degree of sample homogeneity
to obtain significant results. Nevertheless, mass spectrometry can
be applied to identify and quantify proteins from different samples.
In microproteomics, much smaller amounts, in the attomole or
even zeptomole range, are required to sequence a peptide [8].
Cellular protrusions are involved in several biological functions,
such as cell migration, neurite outgrowth, phagocytosis, and cellto-cell communication. More recently, these structures have also
been related to cancer-cell invasion, intercellular transmission of
misfolded proteins in degenerative diseases, and intercellular spread
of infectious pathogens. These physiological and pathological processes are conducted by different types of cellular protrusions, such
as lamellipodia, pseudopodia, filopodia, growth cone, tunneling
nanotubes, and others [9–17]. However, there are many unanswered questions related to their mechanism of formation, their
structural components, or signaling pathways [18]. One of the
principal reasons for the lack of knowledge regarding cellular protrusions is the limited techniques that allow studying them specifically. Previous attempts have been made to isolate cellular
protrusions by using a Boyden chamber [19–21] or an excimer
laser [22]. However, none of these methods are specific since they
do not visualize what they are isolating and they were only able to
enrich pseudopodia, a specific subtype of cellular protrusion, but
could not specifically isolate different types of cellular protrusions.
Here, we describe a protocol that combines two techniques to
enhance our knowledge of the composition of these structures:
LCM, to get an enrichment of cellular protrusions based on morphological features, and MS, to uncover the proteomes of these
structures.
The first roadblock in using LCM to isolate cellular protrusions
is their transient nature and structural fragility. Thus, we first
needed to identify fixative procedures that would be strong enough
to maintain the structural integrity of the cellular protrusions for
laser microdissection but also compatible with downstream
26
Ana Gordon and Karine Gousset
neurons affected by Parkinson or Alzheimer diseases, virus-infected
cells, etc. [2–4]. Moreover, LCM was shown to be able to separate
cellular organelles from specific cells [5], growth cones [6], and
invadopodia [7]. One advantage of using LCM to enrich a sample
with the cells of interest is the possibility of performing subsequent
downstream studies of DNA, RNA, or protein composition.
Proteomics, the study of proteins, is one of the most important
technologies used to acquire new insights into cellular function
since proteins are the primary effectors of all biological activity.
However, the feasibility of combining proteomic techniques with
LCM depends primarily on the sensitivity of the technique used, as
the amount of protein retrieved from the dissected sample can be
very small. Traditional proteomic techniques cannot solve this
problem since they require a high degree of sample homogeneity
to obtain significant results. Nevertheless, mass spectrometry can
be applied to identify and quantify proteins from different samples.
In microproteomics, much smaller amounts, in the attomole or
even zeptomole range, are required to sequence a peptide [8].
Cellular protrusions are involved in several biological functions,
such as cell migration, neurite outgrowth, phagocytosis, and cellto-cell communication. More recently, these structures have also
been related to cancer-cell invasion, intercellular transmission of
misfolded proteins in degenerative diseases, and intercellular spread
of infectious pathogens. These physiological and pathological processes are conducted by different types of cellular protrusions, such
as lamellipodia, pseudopodia, filopodia, growth cone, tunneling
nanotubes, and others [9–17]. However, there are many unanswered questions related to their mechanism of formation, their
structural components, or signaling pathways [18]. One of the
principal reasons for the lack of knowledge regarding cellular protrusions is the limited techniques that allow studying them specifically. Previous attempts have been made to isolate cellular
protrusions by using a Boyden chamber [19–21] or an excimer
laser [22]. However, none of these methods are specific since they
do not visualize what they are isolating and they were only able to
enrich pseudopodia, a specific subtype of cellular protrusion, but
could not specifically isolate different types of cellular protrusions.
Here, we describe a protocol that combines two techniques to
enhance our knowledge of the composition of these structures:
LCM, to get an enrichment of cellular protrusions based on morphological features, and MS, to uncover the proteomes of these
structures.
The first roadblock in using LCM to isolate cellular protrusions
is their transient nature and structural fragility. Thus, we first
needed to identify fixative procedures that would be strong enough
to maintain the structural integrity of the cellular protrusions for
laser microdissection but also compatible with downstream
26
Ana Gordon and Karine Gousset